UspA2 nucleic acid of moraxella catarrhalis

ABSTRACT

The present invention discloses the existence of two novel proteins UspA1 and UspA2, and their respective genes uspA1 and uspA2. Each protein encompasses a region that is conserved between the two proteins and comprises an epitope that is recognized by the MAb 17C7. One or more than one of these species may aggregate to form the very high molecular weight form (i.e. greater than 200 kDa) of the UspA antigen. Compositions and both diagnostic and therapeutic methods for the treatment and study of  M. catarrhalis  are disclosed.

The present application is a Divisional Application of U.S. patentapplication Ser. No. 10/872,769 filed on Jun. 21, 2004 which issued asU.S. Pat. No. 7,344,724 on Jun. 21, 2004, which is a Divisional of Ser.No. 09/336,447 filed Jun. 21, 1999, which issued as U.S. Pat. No.6,310,190, on Oct. 30, 2001, which is a Continuation of InternationalApplication No. PCT/US97/23930, filed Dec. 19, 1997, which claimedpriority to U.S. Provisional Application Ser. No. 60/033,598 filed Dec.20, 1996. The entire text of the above-referenced disclosures arespecifically incorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of microbiology,and clinical bacteriology. More particularly, it concerns sequences ofthe uspA1 and uspA2 genes which encode the proteins UspA1 and UspA2,respectively, both of which encode an epitope reactive with monoclonalantibody (MAb) 17C7 and provide useful epitopes for immunodiagnosis andimmunoprophylaxis.

II. Description of Related Art

It was previously thought that Moraxella catarrhalis, previously knownas Branhamella catarrhalis or Neisseria catarrhalis, was a harmlesssaprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990).However, during the previous decade, it has been determined that thisorganism is an important human pathogen. Indeed, it has been establishedthat this Gram-negative diplococcus is the cause of a number of humaninfections (Murphy, 1989). M. catarrhalis is now known to be the thirdmost common cause of both acute and chronic otitis media (Catlin, 1990;Faden et al., 1990; 1991; Marchant, 1990), the most common disease forwhich infants and children receive health care according to the 1989Consensus Report. This organism also causes acute maxillary sinusitis,generalized infections of the lower respiratory tract (Murphy and Loeb,1989) and is an important cause of bronchopulmonary infections inpatients with underlying chronic lung disease and, less frequently, ofsystemic infections in immunocompromised patients (Melendez and Johnson,1990; Sarubbi et al., 1990; Schonheyder and Ejlertsen, 1989; Wright andWallace, 1989).

The 1989 Consensus Report further concluded that prevention of otitismedia is an important health care goal due to both its occurrence ininfants and children, as well as certain populations of all age groups.In fact, the total financial burden of otitis media has been estimatedto be at least $2.5 billion annually. Vaccines were identified as themost desired approach to prevent this disease for a number of reasons.For example, it was estimated that if vaccines could reduce theincidence of otitis media by 30%, then, the annual health care savingswould be at least $400 million. However, while some progress has beenmade in the development of vaccines for 2 of the 3 common otitis mediapathogens, Streptococcus pneumoniae and Haemophilus influenzae, there isno indication that similar progress has been made with respect to M.catarrhalis. This is particularly troublesome in that M. catarrhalis nowaccounts for approximately 17-20% of all otitis media infection (Murphy,1989). In addition, M. catarrhalis is also a significant cause ofsinusitis (van Cauwenberge et al., 1993) and persistent cough (Gottfarband Brauner, 1994) in children. In the elderly, it infects patients withpredisposing conditions such as chronic-obstructive pulmonary disease(COPD) and other chronic cardiopulmonary conditions (Boyle et al., 1991;Davies and Maesen, 1988; Hager et al., 1987).

Despite its recognized virulence potential, little is known about themechanisms employed by M. catarrhalis in the production of disease orabout host factors governing immunity to this pathogen. An antibodyresponse to M. catarrhalis otitis media has been documented by means ofan ELISA system using whole M. catarrhalis cells as antigen and acuteand convalescent sera or middle ear fluid as the source of antibody(Leinonen et al., 1981). The development of serum bactericidal antibodyduring M. catarrhalis infection in adults was shown to be dependent onthe classical complement pathway (Chapman et al., 1985). And morerecently, it was reported that young children with M. catarrhalis otitismedia develop an antibody response in the middle ear but fail to developa systemic antibody response in a uniform manner (Faden et al., 1992).

Previous attempts have been made to identify and characterize M.catarrhalis antigens that would serve as potentially important targetsof the human immune response to infection (Murphy, 1989; Goldblatt etal., 1990; Murphy et al., 1990). Generally speaking, the surface of M.catarrhalis is composed of outer membrane proteins (OMPs),lipooligosaccharide (LOS) and fimbriae. M. catarrhalis appears to besomewhat distinct from other Gram-negative bacteria in that attempts toisolate the outer membrane of this organism using detergentfractionation of cell envelopes has generally proven to be unsuccessfulin that the procedures did not yield consistent results (Murphy, 1989;Murphy and Loeb, 1989). Moreover, preparations were found to becontaminated with cytoplasmic membranes, suggesting an unusualcharacteristic of the M. catarrhalis cell envelope.

Passive immunization with polyclonal antisera raised against outermembrane vesicles of the M. catarrhalis strain 035E was also found toprotect against pulmonary challenge by the heterologous M. catarrhalisstrain TTA24. In addition, active immunization with M. catarrhalis outermembrane vesicles resulted in enhanced clearance of this organism fromthe lungs after challenge. The positive effect of immunization inpulmonary clearance indicates that antibodies play a major role inimmunoprotection from this pathogen. In addition, the protectionobserved against pulmonary challenge with a heterologous M. catarrhalisstrain demonstrates that one or more conserved surface antigens aretargets for antibodies which function to enhance clearance of M.catarrhalis from the lungs.

Outer membrane proteins (OMPs) constitute major antigenic determinantsof this unencapsulated organism (Bartos and Murphy, 1988) and differentstrains share remarkably similar OMP profiles (Bartos and Murphy, 1988;Murphy and Bartos, 1989). At least three different surface-exposed outermembrane antigens have been shown to be well-conserved among M.catarrhalis strains; these include the 81 kDa CopB OMP (Helminen et al.,1993b), the heat-modifiable CD OMP (Murphy et al., 1993) and thehigh-molecular weight UspA antigen (Helminen et al., 1994). Of thesethree antigens, both the CopB protein and UspA antigen have been shownto bind antibodies which exert biological activity against M.catarrhalis in an animal model (Helminen et al., 1994; Murphy et al.,1993).

The MAb, designated 17C7, was described as binding to UspA, a very highmolecular weight protein that migrated with an apparent molecular weight(in SDS-PAGE) of at least 250 kDa (Helminen et al., 1994; Klingman andMurphy, 1994). MAb 17C7 enhanced pulmonary clearance of M. catarrhalisfrom the lungs of mice when used in passive immunization studies and, incolony blot radioimmunoassay analysis, bound to every isolate of M.catarrhalis examined. This same MAb also reacted, although lessintensely, with another antigen band of approximately 100 kDa, asdescribed in U.S. Pat. No. 5,552,146 (incorporated herein by reference).A recombinant bacteriophage that contained a fragment of M. catarrhalischromosomal DNA that expressed a protein product that bound MAb 17C7 wasalso identified and migrated at a rate similar or indistinguishable fromthat of the native UspA antigen from M. catarrhalis (Helminen et al.,1994).

With the rising importance of this pathogen in respiratory tractinfections, identification of the surface components of this bacteriuminvolved in virulence expression and immunity is becoming moreimportant. To date, there are no vaccines available, against any otherOMP, LOS or fimbriae, that induce protective antibodies against M.catarrhalis. Thus, it is clear that there remains a need to identify andcharacterize useful antigens and which can be employed in thepreparation of immunoprophylactic reagents. Additionally, once such anantigen or antigens is identified, there is a need for providing methodsand compositions which will allow the preparation of vaccines and inquantities that will allow their use on a wide scale basis inprophylactic protocols.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide newUspA1 and UspA2 proteins and genes coding therefor. It also is an objectof the present invention to provide methods of using these new proteins,for example, in the preparation of agents for the treatment andinhibition of M. catarrhalis infection. It also is contemplated thatthrough the use of other technologies such as antibody treatment andimmunoprophylaxis that one can inhibit or even prevent M. catarrhalisinfections.

In satisfying these goals, there are provided epitopic core sequences ofUspA1 and UspA2 which can serve as the basis for the preparation oftherapeutic or prophylactic compositions or vaccines which comprisepeptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length thatelicit an antigenic reaction and a pharmaceutically acceptable buffer ordiluent. These peptides may be coupled to a carrier, adjuvant, anotherpeptide or other molecule such that an effective antigenic response toM. catarrhalis is retained or even enhanced. Alternatively, thesepeptides may act as carriers themselves when coupled to another peptideor other molecule that elicits an antigenic response to M. catarrhalisor another pathogen. For example, UspA2 can serve as a carrier for anoligosaccharide.

In one embodiment, the epitopic core sequences of UspA1 and UspA2comprise one or more isolated peptides of 7, 10, 20, 30, 40, 50 or even60 amino acids in length having the amino acid sequence AQQQDQH (SEQ IDNO:17).

In another embodiment, there are provided nucleic acids, uspA1 anduspA2, which encode the UspA1 and the UspA2 antigens, respectively, aswell as the amino acid sequences of the UspA1 and UspA2 antigens of theM. catarrhalis isolates O35E, TTA24, TTA37, and O46E. It is envisionedthat nucleic acid segments and fragments of the genes uspA1 and uspA2and the UspA1 and UspA2 antigens will be of value in the preparation anduse of therapeutic or prophylactic compositions or vaccines fortreating, inhibiting or even preventing M. catarrhalis infections.

In another embodiment, there is provided a method for inducing an immuneresponse in a mammal comprising the step of providing to the mammal anantigenic composition that comprises an isolated peptide of about 20 toabout 60 amino acids that contains the identified epitopic core sequenceand a pharmaceutically acceptable buffer or diluent.

In another embodiment, there is provided a method for diagnosing M.catarrhalis infection which comprises the step of determining thepresence, in a sample, of an M. catarrhalis amino acid sequencecorresponding to residues of the epitopic core sequences of either theUspA1 or UspA2 antigen. This method may comprise PCR™ detection of thenucleotide sequences or alternatively an immunologic reactivity of anantibody to either a UspA1 or UspA2 antigen.

In a further embodiment, there is provided a method for treating anindividual having an M. catarrhalis infection which comprises providingto the individual an isolated peptide of about 20 to about 60 aminoacids that comprises at least about 10 consecutive residues of the aminoacid sequence identified as an epitopic core sequence of UspA1 or UspA2.

In a still further embodiment, there is provided a method for preventingor limiting an M. catarrhalis infection that comprises providing to asubject an antibody that reacts immunologically with the identifiedepitopic core region of either UspA1 or UspA2 of M. catarrhalis.

In another embodiment, there is provided a method for screening apeptide for reactivity with an antibody that binds immunologically toUspA1, UspA2 or both which comprises the steps of providing the peptideand contacting the peptide with the antibody and then determining thebinding of the antibody to the peptide. This method may comprise animmunoassay such as a western blot, an ELISA, an RIA or animmunoaffinity separation.

In a still further embodiment, there is provided a method for screeninga UspA1 or UspA2 peptide for its ability to induce a protective immuneresponse against M. catarrhalis by providing the peptide, administeringit in a suitable form to an experimental animal, challenging the animalwith M. catarrhalis and then assaying for an M. catarrhalis infection inthe animal. It is envisioned that the animal used will be a mouse thatis challenged by a pulmonary exposure to M. catarrhalis and that theassaying comprises assessing the degree of pulmonary clearance by themouse.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Southern blot analysis of PvuII-digested chromosomal DNA fromstrains of M. catarrhalis using a probe from the uspA1 gene. Bacterialstrain designations are at the top; kilobase (kb) position markers areon the left.

FIG. 2A. Proteins present in whole cell lysates of the wild-type strainO35E and the isogenic uspA1 mutant strain. These proteins were resolvedby SDS-PAGE and stained with Coomassie blue. The left lane (WT) containsthe wild-type strain and right lane (MUT) contains the mutant. Thearrows indicate the protein, approximately 120 kDa in size, that ispresent in the wild-type and missing in the mutant. Kilodalton positionmarkers are on the left.

FIG. 2B. Western blot analysis of whole cell lysates of the wild-typestrain O35E and the isogenic uspA1 mutant strain. These proteins wereresolved by SDS-PAGE and probed with MAb 17C7 in western blot analysis.The left lane (WT) contains the wild-type strain and the right lane(MUT) contains the mutant. Kilodalton position markers are on the left.It can been seen that both strains possess the very high molecularweight band reactive with MAb 17C7 whereas only the wild-type strainalso has a band of approximately 120 kDa that binds this MAb.

FIG. 2C. Western blot analysis of whole cell lysate (WCL) andEDTA-extracted outer membrane vesicles (OMV) from the wild-type strainO35E (WT) and the isogenic uspA1 mutant (MUT) using MAb 17C7. Sampleswere either heated at 37° C. for 15 minutes (H) or at 100° C. for 5minutes (B) prior to SDS-PAGE. Molecular weight position markers (inkilodaltons) are indicated on the left. The open arrow indicates theposition of the very high molecular weight form of the MAb 17C7-reactiveantigen; the closed arrow indicates the position of the approximately120 kDa protein; the open circle indicates the position of theapproximately 70-80 kDa protein.

FIG. 3. Southern blot analysis of chromosomal DNA from the wild-type M.catarrhalis strain O35E and the isogenic uspA1 mutant Chromosomal DNAwas digested with PvuII and probed with a 0.6 kb BglII-PvuII fragmentfrom the THAI gene. The wild-type strain is listed as O35E at the top ofthis figure and the mutant strain is listed as O35E-uspA1⁻. Kilobaseposition markers are present on the left side.

FIG. 4. Western blot reactivity of proteins in M. catarrhalis strainO35E outer membrane vesicles (labeled O35E OMV) and the MF-4-1 GSTfusion protein (labeled GST fusion protein) with MAb 17C7.

FIG. 5. PCR™ products obtained by the use of the T3 and P10 primers(middle lane—0.9 kb product) and the T7 and P9 primers (right lane—1.7kb product) when used in a PCR™ amplification with chromosomal DNA fromthe uspA1 mutant. A kb ladder is present in the first lane; several kbposition markers are listed on the left side of this figure.

FIG. 6A-6C. SDS-PAGE and westerns of purified proteins. FIG. 6A.Coomassie blue stained gel of purified UspA2 (lane 2). FIG. 6B.Coomassie blue stained gel of purified UspA1 prepared without beating ofsample (lane 4), heated for 3 min at 100° C. (lane 5), heated for 5 minat 100° C. (lane 6), and heated for 10 min at 100° C. (lane 7). FIG. 6C.Western of the purified UspA2 (lane 9) and purified UspA1 (lane 10)probed with the 17C7 MAb. Both proteins were heated 10 min. Themolecular size markers in lanes 1, 3, and 8 are as indicated inkilodaltons.

FIG. 7. Interaction of purified UspA1 and UspA2 with HEp-2 cells asdetermined by ELISA. HEp-2 cell monolayers cultured in 96-well platewere incubated with serially diluted UspA1 or UspA2. O35E bacterialstrain was used as the positive control. The bacteria were dilutedanalogous to the proteins beginning with a suspension with an A₅₅₀ of1.0. The bound proteins or attached bacteria were detected with a 1:1mixed antisera to UspA1 and UspA2 as described in the methods.

FIG. 8. Interaction with fibronectin and vitronectin determined by dotblot. The bound vitronectin was detected with rabbit polyclonalantibodies, the protein bound to the fibronectin was detected withpooled sera made against the UspA1 and UspA2

FIG. 9A-9F. The levels of antibodies to the protein UspA1, UspA2 and M.catarrhalis O35E strain in normal human sera. Data are the logytransformed end-point titers of the IgG (FIGS. 9A-9C) and IgA FIGS.9D-9F) antibodies determined by ELISA. The individual titers wereplotted according to age group and the geometric mean titer for each agegroup linked by a solid line. Sera for the 2-18 month old children wereconsecutive samples from a group of ten children.

FIG. 10A-10B. Subclass distribution of IgG antibodies to UspA1 and UspA2in normal human sera FIG. 10A shows titers toward UspA1 and FIG. 10Bshows titers to UspA2.

FIG. 11A-11B. Relationship of serum IgG titers to UspA1 (FIG. 11A) andUspA2 (FIG. 11B) with the bactericidal liter against the O35E straindetermined by logistic regression p<0.05). The solid line indicates thelinear relationship between the IgG titer and bactericidal titer. Brokenlines represent the 95% confidence intervals of the linear fit.

FIG. 12. Schematic drawing showing the relative positions ofdecapeptides 10-24 within the region of UspA1 and UspA2 which binds toMAb 17C7.

FIG. 13. Western dot blot analysis demonstrating reactivity ofdecapeptides 10-24 with MAb 17C7.

FIG. 14A-14B. Partial restriction enzyme map of the uspA1 (FIG. 14A) anduspA2 (FIG. 14B) genes from M. catarrhalis strain O35E and the mutatedversions of these genes. The shaded boxes indicate the open readingframe of each gene. Relevant restriction sites are indicated. PCR™primer sites (P1-P6) are indicated by arrows. The DNA fragmentscontaining the partial uspA1 and uspA2 open reading frames that werederived from M. catarrhalis strain O35E chromosomal DNA by PCR™ andcloned into pBluescriptII SK+ are indicated by black bars. Dotted linesconnect corresponding restriction sites on these DNA inserts and thechromosome. Open bars indicate the location of the kanamycin orchloramphenicol cassettes, respectively. The DNA probes specific foruspA1 or uspA2 are indicated by the appropriate cross-hatched bars andwere amplified by PCR™ from M. catarrhalis strain O35E chromosomal DNAby the use of the oligonucleotide primer pairs

P3 (5′-GACGCTCAACAGCACTAATACG-3′)/ (SEQ ID NO:20) P4(5′-CCAAGCTGATATCACTACC-3′) (SEQ ID NO:21) and P5(5′-TCAATGCCTTTGATGGTC-3′)/ (SEQ ID NO:22) P6(5′-TGTATGCCGCTACTCGCAGCT-3′), (SEQ ID NO:23) respectively.

FIG. 15A-15B. Detection of the UspA1 and UspA2 proteins in wild-type andmutant stains of M. catarrhalis O35E. Proteins present in EDTA-extractedouter membrane vesicles from the wild-type strain (lane 1), the uspA1mutant strain O35E.1 (lane 2), the uspA2 mutant stain O35E.2 (lane 3),and the isogenicuspA1 uspA2 double mutant strain O35E.12 (lane 4) wereresolved by SDS-PAGE, and either stained with Coomassie blue (FIG. 15A)or transferred to nitrocellulose and probed with MAb 17C7 followed byradioiodinated goat anti-mouse immunoglobulin in western blot analysis.In FIG. 15A, the closed arrow indicates the very high molecular weightform of the UspA antigen which is comprised of both UspA1 and UspA2. InFIG. 15B, the bracket on the left indicates the very high molecularweight forms of the UspA1 and UspA2 proteins that bind MAb 17C7. Theopen arrow indicates the 120 kDa, putative monomeric form of UspA1. Theclosed arrow indicates the 85 kDa, putative-monomeric form of UspA2.Molecular weight position markers (in kilodaltons) are present on theleft.

FIG. 16. Comparison of the rate and extent of growth of the wild-typeand mutant strains of M. catarrhalis. The wild-type strain O35E (closedsquares), the uspA1 mutant O35E.1 (open squares), the uspA2 mutantO35E.2 (closed circles), and the uspA1 uspA2 double mutant O35E.12 (opencircles) of M. catarrhalis O35E from overnight broth cultures werediluted to a density of 35 Klett units in BHI broth and subsequentlyallowed to grow at 37° with shaking. Growth was followed by means ofturbidity measurements.

FIG. 17. Susceptibility of wild-type and mutant strains of M.catarrhalis to killing by normal human serum. Cells of the wild-typeparent strain O35E (diamonds), uspA1 mutant O35E.1 (triangles), uspA2mutant O35E.2 (circles), and uspA1 uspA2 double mutant O35E.12 (squares)from logarithmic-phase BHI broth cultures were incubated in the presenceof 10% (v/v) normal human serum (closed symbols) or heat-inactivatednormal human serum (open symbols). Data are presented as the percentageof the original inoculum remaining at each time point.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to the identification of epitopes usefulfor developing potential vaccines against M. catarrhalis. Early work wasdirected at determining the molecular nature of the UspA antigen andcharacterize the epitope which is recognized by the MAb 17C7.Preliminary work indicated that MAb 17C7 recognizes a single antigenicepitope and it was believed that this epitope was encoded by a singlegene. However, isolation of the protein which contained the epitopeyielded unexpected results. MAb 17C7 recognized a single epitope, butthe characteristics of the protein associated with the epitope suggestedthe existence of not one but two separate proteins. Further carefulanalyses led to a surprising discovery. A single epitope of the UspAantigen is recognized by the MAb 17C7, but this epitope is present intwo different proteins, UspA1 and UspA2, which are encoded by twodifferent genes uspA1 and uspA2, respectively, and only have 43%identity to each other. The present invention provides the nucleotidesequences of the genes uspA1 and uspA2, their respective proteinproducts, UspA1 and UspA2, and the shared epitope recognized by MAb17C7.

In addition, the present invention provides insights into the antigenicstructure of the UspA protein based on the analysis of the sequences ofthe UspA1 and UspA2 proteins which comprise the protein.Characterization of the epitopic region of the molecule that is targetedby the MAb 17C7 permits the development of agents that will be useful inprotecting against M. catarrhalis infections, e.g., in the preparationof prophylactic reagents. Particular embodiments relate to the aminoacid and nucleic acids corresponding to the UspA1 and UspA2 proteins,peptides and antigenic compositions derived therefrom, and methods forthe diagnosis and treatment of M. catarrhalis disease.

As stated previously, M. catarrhalis infections present a serious healthchallenge, especially to the young. Thus, there is a clear need todevelop compositions and methods that will aid in the treatment anddiagnosis of this disease. The present invention, by virtue of newinformation regarding the structure of the UspA antigen of M.catarrhalis, and discovery of the two new and distinct proteins UspA1and UspA2 provides such improved compositions and methods. UspA1 andUspA2 represent important antigenic determinants, as the MAb 17C7 hasbeen shown to protect experimental animals, as measured in a pulmonaryclearance model, when provided in passive immunizations.

In a first embodiment, the present invention provides for theidentification of the proteins UspA1 and UspA2 from M. catarrhalisstrain O35E. The UspA1 protein comprises about 831 amino acid residuesand has a predicted mass of about 88,271 daltons (SEQ ID NO:1). TheUspA2 protein comprises about 576 residues and has a predicted mass ofabout 62,483 daltons (SEQ ID NO:3). UspA2 is not a truncated orprocessed form of UspA1.

In a second embodiment, the present invention has identified thespecific epitope to which MAb 17C7 binds. A common peptide sequence,designated as the “3Q” peptide, found between amino acid residues480-502 and 582-604 of the UspA1 protein (SEQ ID NO:1) and residues355-377 of the UspA2 protein (SEQ ID NO:3) of M. catarrhalis strainO35E, encompasses the region which appears to be recognized by MAb 17C7.(Note that numbering of the amino acid residues is based upon strainO35E as provided in SEQ ID NO:3.) It is envisioned that this regionplays an important role in the biology of the pathogen and, from thisinformation, one will deduce amino acids residues that are critical inMAb 17C7 antibody binding. It also is envisioned that, based upon thisinformation, one will be able to design epitopic regions that haveeither a higher or lower affinity for the MAb 17C7 or other antibodies.Further embodiments of the present invention are discussed below.

In another preferred embodiment, the present invention provides DNAsegments, vectors and the like comprising at least one isolated gene,DNA segment or coding region that encodes a M. catarrhalis UspA1 orUspA2 protein, polypeptide, domain, peptide or any fusion proteinthereof. Herein are provided at least an isolated gene, DNA segment orcoding region that encodes a M. catarrhalis uspA1 gene comprising about2493 base pairs (bp) (SEQ ID NO:2) of strain O35E, about 3381 bp (SEQ IDNO:6) of strain O46E, about 3538 bp (SEQ ID NO:10) of strain TTA24, orabout 3292 bp (SEQ ID NO:14) of strain TTA37. Further provided are atleast an isolated gene, DNA segment or coding region that encodes a M.catarrhalis uspA2 gene comprising about 1728 bp (SEQ ID NO:4) of strainO35E, about 3295 bp (SEQ ID NO:8) of strain O46E, about 2673 bp (SEQ IDNO:12), or about 4228 bp (SEQ ID NO:16) of strain TTA37. It isenvisioned that the uspA1 and uspA2 genes will be useful in thepreparation of proteins, antibodies, screening assays for potentialcandidate drugs and the like to treat or inhibit, or even prevent, M.catarrhalis infections.

The present invention also provides for the use of the UspA1 or UspA2proteins or peptides as immunogenic carriers of other agents which areuseful for the treatment, inhibition or even prevention of otherbacterial, viral or parasitic infections. It is envisioned that eitherthe UspA1 or UspA2 antigen, or portions thereof, will be coupled,bonded, bound, conjugated or chemically-linked to one or more agents vialinkers, polylinkers or derivatized amino acids such that a bispecificor multivalent composition or vaccine which is useful for the treatment,inhibition or even prevention of infection by M. catarrhalis and anotherpathogen(s) is prepared. It is further envisioned that the methods usedin the preparation of these compositions will be familiar to those ofskill in the art and, for example, similar to those used to prepareconjugates to keyhole limpet hemocyannin (KLH) or bovine serum albumin(BSA).

It is important to note that screening methods for diagnosis andprophylaxis are readily available, as set forth below. Thus, the abilityto (i) test peptides, mutant peptides and antibodies for theirreactivity with each other and (ii) test peptides and antibodies for theability to prevent infections in vivo, provide powerful tools to developclinically important reagents.

1.0 UspA PROTEINS, PEPTIDES AND POLYPEPTIDES

The present invention, in one embodiment, encompasses the two newprotein sequences, UspA1 and UspA2, and the peptide sequence AQQQDQH(SEQ ID NO:17) identified as the target epitope of MAb 17C7. Inaddition, inspection of the amino acid sequences of the UspA1 and UspA2proteins from four strains of M. catarrhalis indicated that each proteincontained at least one copy of the peptide YELAQQQDQH (SEQ ID NO:18)which binds Mab 17C7 or, in one instance, a peptide nearly identical andhaving the amino acid sequence YDLAQQQDQH (SEQ ID NO:19).

The peptide (YELAQQQDQH, SEQ ID NO:18) occurs twice in UspA1 from strainO35E at residues 486-495 and 588-597 (SEQ ID NO:1) and once in UspA2from strain O35E at residues 358-367 (SEQ ID NO:3). It occurs once inUspA1 from strain TTA24 at residues 497-506 (SEQ ID NO:9) and twice inUspA2 from strain TTA24 at residues 225-234 and 413-422 (SEQ ID NO:11).The peptide YDLAQQQDQH (SEQ ID NO:19) occurs once in UspA1 from strainO46E at residues 448-457 (SEQ ID NO:5) whereas the peptide YELAQQQDQH(SEQ ID NO:18) occurs once in this same protein at residues 649-658 (SEQID NO:5). The peptide YELAQQQDQH (SEQ ID NO:18) occurs once in UspA2from strain O46E at residues 416-425 (SEQ ID NO:7). The peptideYELAQQQDQH (SEQ ID NO:18) occurs twice in UspA1 from strain TTA37 atresidues 478-487 and 630-639 (SEQ ID NO:13) and twice in UspA2 fromstrain TTA37 at residues 522-531 and 681-690 (SEQ ID NO:15).

Also encompassed in the present invention are hybrid moleculescontaining portions from one UspA protein, for example the UspA1protein, fused with portions of the other UspA protein, in this examplethe UspA2 protein, or fused with other proteins which are useful foridentification, such as kanamycin-resistance, or other purposes in thescreening of potential vaccines or further characterization of the UspA1and UspA2 proteins. For example, one may fuse residues 1-350 of anyUspA1 with residues 351-576 of any UspA2. Alternatively, a fusion couldbe generated with sequences from three, four or even five peptideregions represented in a single UspA antigen. Also encompassed arefragments of the disclosed UspA1 and UspA2 molecules, as well asinsertion, deletion or replacement mutants in which non-UspA sequencesare introduced, UspA sequences are removed, or UspA sequences arereplaced with non-UspA sequences, respectively.

UspA1 and UspA2 proteins, according to the present invention, may beadvantageously cleaved into fragments for use in further structural orfunctional analysis, or in the generation of reagents such asUspA-related polypeptides and UspA-specific antibodies. This can beaccomplished by treating purified or unpurified UspA1 and/or UspA2 witha peptidase such as endoproteinase glu-C (Boehriger, Indianapolis,Ind.). Treatment with CNBr is another method by which UspA1 and/or UspA2fragments may be produced from their natural respective proteins,Recombinant techniques also can be used to produce specific fragments ofUspA1 or UspA2.

More subtle modifications and changes may be made in the structure ofthe encoded UspA1 or UspA2 polypeptides of the present invention andstill obtain a molecule that encodes a protein or peptide withcharacteristics of the natural UspA antigen. The following is adiscussion based upon changing the amino acids of a protein to create anequivalent, or even an improved, second-generation molecule. The aminoacid changes may be achieved by changing the codons of the DNA sequence,according to the following codon table:

TABLE I Amino acid names and abbreviations Codons Alanine Ala A GCA GCCGCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acidGlu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It is known that certain amino acids may be substituted for other aminoacids in a protein structure in order to modify or improve its antigenicor immunogenic activity (see, e.g., Kyte & Doolittle, 1982; Hopp, U.S.Pat. No. 4,554,101, incorporated herein by reference). For example,through the substitution of alternative amino acids, smallconformational changes may be conferred upon a polypeptide which resultin increased activity or stability. Alternatively, amino acidsubstitutions in certain polypeptides may be utilized to provideresidues which may then be linked to other molecules to providepeptide-molecule conjugates which retain enough antigenicity of thestarting peptide to be useful for other purposes. For example, aselected UspA1 or UspA2 peptide bound to a solid support might beconstructed which would have particular advantages in diagnosticembodiments.

The importance of the hydropathic index of amino acids in conferringinteractive biological function on a protein has been discussedgenerally by Kyte & Doolittle (1982), wherein it is found that certainamino acids may be substituted for other amino acids having a similarhydropathic index or core and still retain a similar biologicalactivity. As displayed in Table II below, amino acids are assigned ahydropathic index on the basis of their hydrophobicity and chargecharacteristics. It is believed that the relative hydropathic characterof the amino acid determines the secondary structure of the resultantprotein, which in turn defines the interaction of the protein withsubstrate molecules. Preferred substitutions which result in anantigenically equivalent peptide or protein will generally involve aminoacids having index scores within ±2 units of one another, and morepreferably within ±1 unit, and even more preferably, within ±0.5 units.

TABLE II Amino Acid Hydropathic Index Isoleucine 4.5 Valine 4.2 Leucine3.8 Phenylalanine 2.8 Cysteine/cystine 2.5 Methionine 1.9 Alanine 1.8Glycine −0.4 Threonine −0.7 Tryptophan −0.9 Serine −0.8 Tyrosine −1.3Proline −1.6 Histidine −3.2 Glutamic Acid −3.5 Glutamine −3.5 AsparticAcid −3.5 Asparagine −3.5 Lysine −3.9 Arginine −4.5Thus, for example, isoleucine, which has a hydropathic index of +4.5,will preferably be exchanged with an amino acid such as valine (+4.2) orleucine (+3.8). Alternatively, at the other end of the scale, lysine(−3.9) will preferably be substituted for arginine (−4.5), and so on.

Substitution of like amino acids may also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentprotein or peptide thereby created is intended for use in immunologicalembodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference,states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith its immunogenicity and antigenicity, i.e. with an importantbiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, each amino acid has also beenassigned a hydrophilicity value. These values are detailed below inTable III.

TABLE III Amino Acid Hydrophilic Index arginine +3.0 lysine +3.0aspartate +3.0 ± 1 glutamate +3.0 ± 1 serine +0.3 asparagine +0.2glutamine +0.2 glycine 0 threonine −0.4 alanine −0.5 histidine −0.5proline −0.5 ± 1 cysteine −1.0 methionine −1.3 valine −1.5 leucine −1.8isoleucine −1.8 tyrosine −2.3 phenylalanine −2.5 tryptophan −3.4

It is understood that one amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

Accordingly, these amino acid substitutions are generally based on therelative similarity of R-group substituents, for example, in terms ofsize, electrophilic character, charge, and the like. In general,preferred substitutions which take various of the foregoingcharacteristics into consideration will be known to those of skill inthe art and include, for example, the following combinations: arginineand lysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine.

In addition, peptides derived from these polypeptides, includingpeptides of at least about 6 consecutive amino acids from thesesequences, are contemplated. Alternatively, such peptides may compriseabout 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59or 60 consecutive residues. For example, a peptide that comprises 6consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3to 8 and so on of the UspA1 or UspA2 protein. Such peptides may berepresented by the formulax to (x+n)=5′ to 3′ the positions of the first and last consecutiveresidueswhere x is equal to any number from 1 to the full length of a UspA1 orUspA2 protein and n is equal to the length of the peptide minus 1. So,for UspA1, x=1 to 831, for UspA2, x=1 to 576. Where the peptide is 10residues long (n=10−1), the formula represents every 10-mer possible foreach antigen. For example, where x is equal to 1 the peptide wouldcomprise residues 1 to (1+[10−1]), or 1 to 10. Where x is equal to 2,the peptide would comprise residues 2 to (2+[10−2]), or 2 to 11, and soon.

Syntheses of peptides are readily achieved using conventional synthetictechniques such as the solid phase method (e.g., through the use of acommercially available peptide synthesizer such as an Applied BiosystemsModel 430A Peptide Synthesizer). Peptides synthesized in this manner maythen be aliquoted in predetermined amounts and stored in conventionalmanners, such as in aqueous solutions or, even more preferably, in apowder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may bedesirable to include agents which will inhibit microbial growth, such assodium azide or Merthiolate. For extended storage in an aqueous state itwill be desirable to store the solutions at 4° C., or more preferably,frozen. Of course, where the peptide(s) are stored in a lyophilized orpowdered state, they may be stored virtually indefinitely, e.g., inmetered aliquots that may be rehydrated with a predetermined amount ofwater (preferably distilled, deionized) or buffer prior to use.

Of particular interest are peptides that represent epitopes that liewithin the UspA antigen and are encompassed by the UspA1 and UspA2proteins of the present invention. An “epitope” is a region of amolecule that stimulates a response from a T-cell or B-cell, and hence,elicits an immune response from these cells. An epitopic core sequence,as used herein, is a relatively short stretch of amino acids that isstructurally “complementary” to, and therefore will bind to, bindingsites on antibodies or T-cell receptors. It will be understood that, inthe context of the present disclosure, the term “complementary” refersto amino acids or peptides that exhibit an attractive force towards eachother. Thus, certain epitopic core sequences of the present inventionmay be operationally defined in terms of their ability to compete withor perhaps displace the binding of the corresponding UspA antigen to thecorresponding UspA-directed antisera.

The identification of epitopic core sequences is known to those of skillin the art. For example U.S. Pat. No. 4,554,101 teaches identificationand preparation of epitopes from amino acid sequences on the basis ofhydrophilicity, and by Chou-Fasman analyses. Numerous computer programsare available for use in predicting antigenic portions of proteins,examples of which include those programs based upon Jameson-Wolfanalyses (Jameson and Wolf, 1988; Wolf et al., 1988), the programPepPlot® (Brutlag et al., 1990; Weinberger et al., 1985), and other newprograms for protein tertiary structure prediction (Fetrow & Bryant,1993) that can be used in conjunction with computerized peptide sequenceanalysis programs.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would be on the order ofabout 6 amino acids in length. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

2.0 UspA1 AND UspA2 NUCLEIC ACIDS

In addition to polypeptides, the present invention also encompassesnucleic acids encoding the UspA1 (SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10and SEQ ID NO:14) and UspA2 (SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 andSEQ ID NO:16) proteins from the exemplary M. catarrhalis strains O35E,O46E, TTA24 and TTA37, respectively. Because of the degeneracy of thegenetic code, many other nucleic acids also may encode a given UspA1 orUspA2 protein. For example, four different three-base codons encode theamino acids alanine, glycine, proline, threonine and valine, while sixdifferent codons encode arginine, leucine and serine. Only methionineand tryptophan are encoded by a single codon. Table I provides a list ofamino acids and their corresponding codons for use in such embodiments.In order to generate any nucleic acid encoding UspA1 or UspA2, one needonly refer to the codon table provided herein. Substitution of thenatural codon with any codon encoding the same amino acid will result ina distinct nucleic acid that encodes UspA1 or UspA2. As a practicalmatter, this can be accomplished by site-directed mutagenesis of anexisting uspA1 or uspA2 gene or de novo chemical synthesis of one ormore nucleic acids.

These observations regarding codon selection, site-directed mutagenesisand chemical synthesis apply with equal force to the discussion ofsubstitutional mutant UspA1 or UspA2 peptides and polypeptides, as setforth above. More specifically, substitutional mutants generated bysite-directed changes in the nucleic acid sequence that are designed toalter one or more codons of a given polypeptide or epitope may provide amore convenient way of generating large numbers of mutants in a rapidfashion. The nucleic acids of the present invention provide for a simpleway to generate fragments (e.g., truncations) of UspA1 or UspA2,UspA1-UspA2 fusion molecules (discussed above) and UspA1 or UspA2fusions with other molecules. For example, utilization of restrictionenzymes and nuclease in the uspA1 or uspA2 gene permits one tomanipulate the structure of these genes, and the resulting geneproducts.

The nucleic acid sequence information provided by the present disclosurealso allows for the preparation of relatively short DNA (or RNA)sequences that have the ability to specifically hybridize to genesequences of the selected uspA1 or uspA2 gene. In these aspects nucleicacid probes of an appropriate length are prepared based on aconsideration of the coding sequence of the uspA1 or uspA2 gene, orflanking regions near the uspA1 or uspA2 gene, such as regionsdownstream and upstream in the M. catarrhalis chromosome. The ability ofsuch nucleic acid probes to specifically hybridize to either uspA1 oruspA2 gene sequences lends them particular utility in a variety ofembodiments. For example, the probes can be used in a variety ofdiagnostic assays for detecting the presence of pathogenic organisms ina given sample. In addition, these oligonucleotides can be inserted, inframe, into expression constructs for the purpose of screening thecorresponding peptides for reactivity with existing antibodies or forthe ability to generate diagnostic or therapeutic reagents.

To provide certain of the advantages in accordance with the invention,the preferred nucleic acid sequence employed for hybridization studiesor assays includes sequences that are complementary to at least a 10 to20, or so, nucleotide stretch of the sequence, although sequences of 30to 60 or so nucleotides are also envisioned to be useful. A size of atleast 9 nucleotides in length helps to ensure that the fragment will beof sufficient length to form a duplex molecule that is both stable andselective. Though molecules having complementary sequences overstretches greater than 10 bases in length are generally preferred, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of the specific hybrid moleculesobtained. Thus, one will generally prefer to design nucleic acidmolecules having either uspA1 or uspA2 gene-complementary stretches of15 to 20 nucleotides, or even longer, such as 30 to 60, where desired.Such fragments may be readily prepared by, for example, directlysynthesizing the fragment by chemical means, by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.No. 4,603,102, or by introducing selected sequences into recombinantvectors for recombinant production.

The probes that would be useful may be derived from any portion of thesequences of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16. Therefore,probes are specifically contemplated that comprise nucleotides 1 to 9,or 2 to 10, or 3 to 11 and so forth up to a probe comprising the last 9nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ IDNO:14 or SEQ ID NO:16. Thus, each probe would comprise at least about 9linear nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ IDNO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 orSEQ ID NO:14 or SEQ ID NO:16, designated by the formula “n to n+8,”where n is an integer from 1 to the number of nucleotides in thesequence. Longer probes that hybridize to the uspA1 or uspA2 gene underlow, medium, medium-high and high stringency conditions are alsocontemplated, including those that comprise the entire nucleotidesequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16. Thishypothetical may be repeated for probes having lengths of about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 and greater bases.

In that the UspA antigenic epitopes of the present invention arebelieved to be indicative of pathogenic Moraxella species as exemplifiedby strains O35E, O46E, TTA24 and TTA37, the probes of the presentinvention will find particular utility as the basis for diagnostichybridization assays for detecting UspA1 or UspA2 DNA in clinicalsamples. Exemplary clinical samples that can be used in the diagnosis ofinfections are thus any samples which could possibly include Moraxellanucleic acid, including middle ear fluid, sputum, mucus; bronchoalveolarfluid, amniotic fluid or the like. A variety of hybridization techniquesand systems are known which can be used in connection with thehybridization aspects of the invention, including diagnostic assays suchas those described in Falkow et al., U.S. Pat. No. 4,358,535. Dependingon the application envisioned, one will desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofthe probe toward the target sequence. For applications requiring a highdegree of selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, for example, one will selectrelatively low salt and/or high temperature conditions, such as providedby 0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. These conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent hybridization conditions are calledfor in order to allow formation of the heteroduplex. In thesecircumstances, one would desire to employ conditions such as 0.15M-0.9Msalt, at tempers ranging from 20° C. to 55° C. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and the method of choice will generally depend on the desired results.

In certain embodiments, one may desire to employ nucleic acid probes toisolate variants from clone banks containing mutated clones. Inparticular embodiments, mutant clone colonies growing on solid mediawhich contain variants of the UspA1 and/or UspA2 sequence could beidentified on duplicate filters using hybridization conditions andmethods, such as those used in colony blot assays, to obtainhybridization only between probes containing sequence variants andnucleic acid sequence variants contained in specific colonies. In thismanner, small hybridization probes containing short variant sequences ofeither the uspA1 or uspA2 gene may be utilized to identify those clonesgrowing on solid media which contain sequence variants of the entireuspA1 or uspA2 gene. These clones can then be grown to obtain desiredquantities of the variant UspA1 or UspA2 nucleic acid sequences or thecorresponding UspA antigen.

In clinical diagnostic embodiments, nucleic acid sequences of thepresent invention are used in combination with an appropriate means,such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including radioactive,enzymatic or other ligands, such as avidin/biotin, which are capable ofgiving a detectable signal. In preferred diagnostic embodiments, onewill likely desire to employ an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of radioactive or other environmentalundesirable reagents. In the case of enzyme tags, calorimetric indicatorsubstrates are known which can be employed to provide a means visible tothe human eye or spectrophotometrically, to identify specifichybridization with pathogen nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridizations aswell as in embodiments employing a solid phase. In embodiments involvinga solid phase, the test DNA (or RNA) from suspected clinical samples,such as exudates, body fluids (e.g., amniotic fluid, middle eareffusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed orotherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C contents, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules specific hybridization isdetected, or even quantified, by means of the label.

The nucleic acid sequences which encode for the UspA1 and/or UspA2epitopes, or their variants, may be useful in conjunction with PCR™methodology to detect M. catarrhalis. In general, by applying the PCR™technology as set out, e.g., in U.S. Pat. No. 4,603,102, one may utilizevarious portions of either the uspA1 or uspA2 sequence asoligonucleotide probes for the PCR™ amplification of a defined portionof a uspA1 or uspA2 nucleic acid in a sample. The amplified portion ofthe uspA1 or uspA2 sequence may then be detected by hybridization with ahybridization probe containing a complementary sequence. In this manner,extremely small concentrations of M. catarrhalis nucleic acid maydetected in a sample utilizing uspA1 or uspA2 sequences.

3.0 VECTORS, HOST CELLS AND CULTURES FOR PRODUCING UspA1 AND/OR UspA2ANTIGENS

In order to express a UspA1 and/or UspA2 polypeptide, it is necessary toprovide an uspA1 and/or uspA2 gene in an expression cassette. Theexpression cassette contains a UspA1 and/or UspA2-encoding nucleic acidunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene. Those promoters most commonly used inprokaryotic recombinant DNA construction include the B-lactamase(penicillinase) and lactose promoter systems (Chang et al., 1978;Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp)promoter system (Goeddel et al., 1980; EPO Appl. Publ. No. 0036776).While these are the most commonly used, other microbial promoters havebeen discovered and utilized and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (EPO Appl. Publ. No. 0036776).Additional examples of useful promoters are provided in Table IV below.

TABLE IV Promoters References Immunoglobulin Heavy Chain Hanerji et al.,1983; Gilles et al., 1983; Grosschedl and Baltimore, 1985; Atchinson andPerry, 1986, 1987; Imler et al., 1987; Weinberger et al., 1988;Kiledjian et al., 1988; Porton et al., 1990 Immunoglobulin Light ChainQueen and Baltimore, 1983; Picard and Schaffner, 1984 T-Cell ReceptorLuria et al., 1987, Winoto and Baltimore, 1989; Redondo et al., 1990 HLADQ a and DQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn et al.,1986; Fujita et al., 1987; Goodbourn and Maniatis, 1985 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al., 1989 MuscleCreatine Kinase Jaynes et al., 1988; Horlick and Benfield, 1989; Johnsonet al., 1989a Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein Karin et al., 1987; Culotta andHamer, 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminGene Pinkert et al., 1987, Tronche et al., 1989, 1990 a-FetoproteinGodbout el al., 1988; Campere and Tilghman, 1989 t-Globin Bodine andLey, 1987; Perez-Stable and Constantini, 1990 β-Globin Trudel andConstantini, 1987 e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986;Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell AdhesionMolecule Hirsch et al., 1990 (NCAM) a_(1-Antitrypain) Latimer et al.,1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse or Type I Collagen Ripeet al., 1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 andGRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A(SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989Platelet-Derived Growth Factor Pech et al., 1989 Duchenne MuscularDystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al.,1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr andClarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang andCalame, 1986; Ondek et al., 1987; Kuhl et al., 1987 Schaffner et al.,1988 Polyoma Swartzendruber and Lehman, 1975; Vasseur et al., 1980;Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland Villarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a,b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesenet al., 1988; Celander et al., 1988; Chol et al., 1988; Reisman andRotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan;1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987,Stephens and Hentschel, 1987; Glue et al., 1988 Hepatitis B Virus Bullaand Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987;Spandau and Lee, 1988; Vannice and Levinson, 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber and Cullan, 1988; Jakobovits et al.,1988; Feng and Holland, 1988; Takebe et al., 1988; Rowen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp and Marciniak, 1989;Braddock et al., 1989 Cytomegalovirus Weber et al., 1984; Boshart etal., 1985; Foecking and Hofstetter, 1986 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

The appropriate expression cassette can be inserted into a commerciallyavailable expression vector by standard subcloning techniques. Forexample, the E. coli vectors pUC or pBluescript™ may be used accordingto the present invention to produce recombinant UspA1 and/or UspA2polypeptide in vitro. The manipulation of these vectors is well known inthe art. In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector ordinarily carries areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (Bolivar et al., 1977). pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used as atransforming vector in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making recombinant phage vectorwhich can be used to transform host cells, such as E. coli LE392.

In one embodiment, the UspA antigen is expressed as a fusion protein byusing the pGEX4T-2 protein fusion system (Pharmacia LKB, Piscataway,N.J.), allowing characterization of the UspA antigen as comprising boththe UspA1 and UspA2 proteins. Additional examples of fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverley,Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system(Qiagen, Chatsworth, Calif.). Some of these fusion systems producerecombinant protein bearing only a small number of additional aminoacids, which are unlikely to affect the functional capacity of therecombinant protein. For example, both the FLAG system and the 6×Hissystem add only short sequences, both of which are known to be poorlyantigenic and which do not adversely affect folding of the protein toits native conformation. Other fusion systems produce proteins where itis desirable to excise the fusion partner from the desired protein. Inanother embodiment, the fusion partner is linked to the recombinantprotein by a peptide sequence containing a specific recognition sequencefor a protease. Examples of suitable sequences are those recognized bythe Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.)or Factor Xa (New England Biolabs, Beverley, Mass.).

E. coli is a preferred prokaryotic host. For example, E. coli strain RR1is particularly useful. Other microbial strains which may be usedinclude E. coli strains such as E. coli LE392, E. coli B, and E. coli X1776 (ATCC No. 31537). The aforementioned strains, as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such asBacillus subtilis, or other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcescens, and various Pseudomonas species maybe used. These examples are, of course, intended to be illustrativerather than limiting. Recombinant bacterial cells, for example E. coli,are grown in any of a number of suitable media, for example LB, and theexpression of the recombinant polypeptide induced by adding IPTG to themedia or switching incubation to a higher temperature. After culturingthe bacteria for a further period of between 2 and 24 hours, the cellsare collected by centrifugation and washed to remove residual media. Thebacterial cells are then lysed, for example, by disruption in a cellhomogenizer and centrifuged to separate the dense inclusion bodies andcell membranes from the soluble cell components. This centrifugation canbe performed under conditions whereby the dense inclusion bodies areselectively enriched by incorporation of sugars such as sucrose into thebuffer and centrifugation at a selective speed.

If the recombinant protein is expressed in the inclusion bodies, as isthe case in many instances, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g.8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents such as β-mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate thepolypeptide for several hours under conditions suitable for the proteinto undergo a refolding process into a conformation which more closelyresembles that of the native protein. Such conditions generally includelow protein concentrations less than 500 μg/ml, low levels of reducingagent, concentrations of urea less than 2 M and often the presence ofreagents such as a mixture of reduced and oxidized glutathione whichfacilitate the interchange of disulfide bonds within the proteinmolecule.

The refolding process can be monitored, for example, by SDS-PAGE or withantibodies which are specific for the native molecule (which can beobtained from animals vaccinated with the native molecule isolated frombacteria). Following refolding, the protein can then be purified furtherand separated from the refolding mixture by chromatography on any ofseveral supports including ion exchange resins, gel permeation resins oron a variety of affinity columns.

There are a variety of other eukaryotic vectors that provide a suitablevehicle in which recombinant UspA proteins can be produced. In variousembodiments of the invention, the expression construct may comprise avirus or engineered construct derived from a viral genome. The abilityof certain viruses to enter cells via receptor-mediated endocytosis andto integrate into host cell genome and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (Ridgeway, 1988; Nicolas andRubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The firstviruses used as vectors were DNA viruses including the papovaviruses(simian virus 40 (SV40), bovine papilloma virus, and polyoma) (Ridgeway,1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;Baichwal and Sugden, 1986) and adeno-associated viruses. Retrovirusesalso are attractive gene transfer vehicles (Nicolas and Rubenstein,1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988)adeno-associated virus (Ridgeway, 1988) and herpes simplex virus (HSV)(Glorioso et al., 1995). Such vectors may be used to (i) transform celllines in vitro for the purpose of expressing proteins of interest or(ii) to transform cells in vitro or in vivo to provide therapeuticpolypeptides in a gene therapy scenario.

With respect to eukaryotic vectors, the term promoter will be used hereto refer to a group of transcriptional control modules that areclustered around the initiation site for RNA polymerase II. Much of thethinking about how promoters are organized derives from analyses ofseveral viral promoters, including those for the HSV thymidine kinase(tk) and SV40 early transcription units. These studies, augmented bymore recent work, have shown that promoters are composed of discretefunctional modules, each consisting of approximately 7-20 bp of DNA, andcontaining one or more recognition sites for transcriptional activatoror repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally speaking, such a promotermight include either a human or viral promoter. Preferred promotersinclude those derived from HSV, including the α4 promoter. Anotherpreferred embodiment is the tetracycline controlled promoter.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. Table IV lists severalpromoters which may be employed, in the context of the presentinvention, to regulate the expression of a transgene. This list is notintended to be exhaustive of all the possible elements involved in thepromotion of transgene expression but, merely, to be exemplary thereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization. Table V listsseveral enhancers, of course, this list is not meant to be limiting butexemplary.

TABLE V Enhancer Inducer References MT II Phorbol Ester (TFA) Palmiteret al., 1982; Haslinger and Heavy metals Karin, 1985; Searle et al.,1985; Stuart et al., 1985; Imagawa et al., 1987; Karin  ®, 1987; Angelet al., 1987b; McNeall et al., 1989 MMTV (mouse Glucocorticoids Huang etal., 1981; Lee et al., 1981; mammary tumor Majors and Varmus, 1983;Chandler virus) et al., 1983; Lee et al., 1984; Fonta et al., 1985;Sakai et al., 1986 β-Interferon poly(rI)X Tavernier et al., 1983poly(rc) Adenovirus 5 E2 Ela Imperiale and Nevins, 1984 CollagenasePhorbol Ester (TPA) Angle et al., 1987a Stromelysin Phorbol Ester (TPA)Angle et al., 1987b SV40 Phorbol Ester (TFA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 Resendezet al., 1988 a-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin SerumRittling et al., 1989 MHC Class I Gene Interferon Blanar et al., 1989H-2kb HSP70 Ela, SV40 Large T Taylor et al., 1989; Taylor and AntigenKingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, 1989Tumor Necrosis FMA Hensel et al., 1989 Factor Thyroid Thyroid HormoneChatterjee et al., 1989 Stimulating Hormone a Gene

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression of atransgene. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

Host cells include eukaryotic microbes, such as yeast cultures may alsobe used. Saccharomyces cerevisiae, or common baker's yeast is the mostcommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb et al.,1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmidalready contains the trp1 gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trp1lesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to eukaryotic microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and WI38, BHK, COS-7, 293 and MDCK cell lines.Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

4.0 PREPARATION OF ANTIBODIES TO UspA PROTEINS

Antibodies to UspA1 or UspA2 peptides or polypeptides may be readilyprepared through use of well-known techniques, such as those exemplifiedin U.S. Pat. No. 4,196,265. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., purified or partially purified protein, synthetic protein orfragments thereof, as discussed in the section on vaccines. Animals tobe immunized are mammals such as cats, dogs and horses, although thereis no limitation other than that the subject be capable of mounting animmune response of some kind. The immunizing composition is administeredin a manner effective to stimulate antibody producing cells. Rodentssuch as mice and rats are preferred animals, however, the use of rabbit,sheep or frog cells is possible. The use of rats may provide certainadvantages, but mice are preferred, with the BALB/c mouse being mostpreferred as the most routinely used animal and one that generally givesa higher percentage of stable fusions.

For generation of monoclonal antibodies (MAbs), following immunization,somatic cells with the potential for producing antibodies, specificallyB lymphocytes (B cells), are selected for use in the MAb generatingprotocol. These cells may be obtained from biopsied spleens, tonsils orlymph nodes, or from a peripheral blood sample. Spleen cells andperipheral blood cells are preferred, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because peripheral blood is easily accessible.Often, a panel of animals will have been immunized and the spleen of theanimal with the highest antibody titer removed. Spleen lymphocytes areobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

The antibody-producing B cells from the immunized animal are then fusedwith cells of an immortal myeloma cell line, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells, called“hybridomas.”

Any one of a number of myeloma cells may be used and these are known tothose of skill in the art. For example, where the immunized animal is amouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one mayuse R120.RCY3, Y3-Ag1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with human cellfusions.

One preferred murine myeloma cell line is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. This does not pose a problem, however, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culture in a selective medium. Theselective medium generally is one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby single-clone dilution in microtiter plates, followed by testing theindividual clonal supernatants (after about two to three weeks) for thedesired reactivity. The assay should be sensitive, simple and rapid,such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays,plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected, usually in the peritoneal cavity, into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Monoclonal antibodies of the present invention also includeanti-idiotypic antibodies produced by methods well-known in the artMonoclonal antibodies according to the present invention also may bemonoclonal heteroconjugates, i.e., hybrids of two or more antibodymolecules. In another embodiment, monoclonal antibodies according to theinvention are chimeric monoclonal antibodies. In one approach, thechimeric monoclonal antibody is engineered by cloning recombinant DNAcontaining the promoter, leader, and variable-region sequences from amouse antibody producing cell and the constant-region exons from a humanantibody gene. The antibody encoded by such a recombinant gene is amouse-human chimera. Its antibody specificity is determined by thevariable region derived from mouse sequences. Its isotype, which isdetermined by the constant region, is derived from human DNA.

In another embodiment, the monoclonal antibody according to the presentinvention is a “humanized” monoclonal antibody, produced by techniqueswell-known in the art. That is, mouse complementary determining regions(“CDRs”) are transferred from heavy and light V-chains of the mouse Iginto a human V-domain, followed by the replacement of some humanresidues in the framework regions of their murine counterparts.“Humanized” monoclonal antibodies in accordance with this invention areespecially suitable for use in in vivo diagnostic and therapeuticmethods for treating Moraxella infections.

As stated above, the monoclonal antibodies and fragments thereofaccording to this invention can be multiplied according to in vitro andin vivo methods well-known in the art. Multiplication in vitro iscarried out in suitable culture media such as Dulbecco's modified Eaglemedium or RPMI 1640 medium, optionally replenished by a mammalian serumsuch as fetal calf serum or trace elements and growth-sustainingsupplements, e.g., feeder cells, such as normal mouse peritoneal exudatecells, spleen cells, bone marrow macrophages or the like. In vitroproduction provides relatively pure antibody preparations and allowsscale-up to give large amounts of the desired antibodies. Techniques forlarge scale hybridoma cultivation under tissue culture conditions areknown in the art and include homogenous suspension culture, e.g., in anairlift reactor or in a continuous stirrer reactor or immobilized orentrapped cell culture.

Large amounts of the monoclonal antibody of the present invention alsomay be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals which are histocompatible with the parent cells,e.g., syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as Pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonalantibody of the invention can be obtained from monoclonal antibodiesproduced as described above, by methods which include digestion withenzymes such as pepsin or papain and/or cleavage of disulfide bonds bychemical reduction. Alternatively, monoclonal antibody fragmentsencompassed by the present invention can be synthesized using anautomated peptide synthesizer, or they may be produced manually usingtechniques well known in the art.

The monoclonal conjugates of the present invention are prepared bymethods known in the art, e.g., by reacting a monoclonal antibodyprepared as described above with, for instance, an enzyme in thepresence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents, or by reaction with an isothiocyanate. Conjugateswith metal chelates are similarly produced. Other moieties to whichantibodies may be conjugated include radionuclides such as ³H, ¹²⁵I,¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, and⁹⁹mTc, are other useful labels which can be conjugated to antibodies.Radio-labeled monoclonal antibodies of the present invention areproduced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Monoclonal antibodies according to the invention may be labeled withtechnetium-⁹⁹m by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody.

5.0 USE OF PEPTIDES AND MONOCLONAL ANTIBODIES IN IMMUNOASSAYS

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and western blot methods, as well as other procedures which mayutilize antibodies specific to CopB epitopes. While ELISAs arepreferred, it will be readily appreciated that such assays include RIAsand other non-enzyme linked antibody binding assays or procedures.Additionally, it is proposed that monoclonal antibodies specific to theparticular UspA epitope may be utilized in other useful applications.For example, their use in immunoabsorbent protocols may be useful inpurifying native or recombinant UspA proteins or variants thereof.

It also is proposed that the disclosed UspA1 and UspA2 peptides of theinvention will find use as antigens for raising antibodies and inimmunoassays for the detection of anti-UspA antigen-reactive antibodies.In a variation on this embodiment, UspA1 and UspA2 mutant peptides maybe screened, in immunoassay format, for reactivity against UspA1- orUspA2-specific antibodies, such as MAb 17C7. In this way, a mutationalanalysis of various epitopes may be performed. Results from suchanalyses may then be used to determine which additional UspA1 or UspA2epitopes may be recognized by antibodies and useful in the preparationof potential vaccines for Moraxella.

Diagnostic immunoassays include direct culturing of bodily fluids,either in liquid culture or on a solid support such as nutrient agar. Atypical assay involves collecting a sample of bodily fluid from apatient and placing the sample in conditions optimum for growth of thepathogen. The determination can then be made as to whether the microbeexists in the sample. Further analysis can be carried out to determinethe hemolyzing properties of the microbe.

Immunoassays encompassed by the present invention include, but are notlimited to those described in U.S. Pat. No. 4,367,110 (double monoclonalantibody sandwich assay) and U.S. Pat. No. 4,452,901 (western blot).Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in-vitro and in vivo.

Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the anti-UspA antibodies of the invention areimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a test compositionsuspected of containing the desired antigen, such as a clinical sample,is added to the wells. After binding and washing to removenon-specifically bound immune complexes, the bound antigen may bedetected. Detection is generally achieved by the addition of anotherantibody, specific for the desired antigen, that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection may also be achieved by the addition of a second antibodyspecific for the desired antigen, followed by the addition of a thirdantibody that has binding affinity for the second antibody, with thethird antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the UspAantigen are immobilized onto the well surface and then contacted withthe anti-UspA antibodies. After binding and appropriate washing, thebound immune complexes are detected. Where the initial antigen specificantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antigen specificantibody, with the second antibody being linked to a detectable label.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the primary antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if desired.

Competition ELISAs are also possible in which test samples compete forbinding with known amounts of labeled antigens or antibodies. The amountof reactive species in the unknown sample is determined by mixing thesample with the known labeled species before or during incubation withcoated wells. (Antigen or antibodies may also be linked to a solidsupport, such as in the form of beads, dipstick, membrane or columnmatrix, and the sample to be analyzed applied to the immobilized antigenor antibody.) The presence of reactive species in the sample acts toreduce the amount of labeled species available for binding to the welland thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of 25° to27° C. Following incubation, the antisera-contacted surface is washed soas to remove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. Of course, inthat the test sample will typically be of human origin, the secondantibody will preferably be an antibody having specificity in generalfor human IgG. To provide a detecting means, the second antibody willpreferably have an associated enzyme that will generate a colordevelopment upon incubating with an appropriate chromogenic substrate.Thus, for example, one will desire to contact and incubate theantisera-bound surface with a urease or peroxidase-conjugated anti-humanIgG for a period of time and under conditions which favor thedevelopment of immunocomplex formation (e.g., incubation for 2 hours atroom temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer. Alternatively, the label maybe a chemiluminescent one. The use of such labels is described in U.S.Pat. Nos. 5,310,687, 5,238,808 and 5,221,605.

6.0 PROPHYLACTIC USE OF UspA PEPTIDES AND UspA-SPECIFIC ANTIBODIES

In a further embodiment of the present invention, there are providedmethods for active and passive immunoprophylaxis. Activeimmunoprophylaxis will be discussed first, followed by a discussion onpassive immunoprophylaxis. It should be noted that the discussion offormulating vaccine compositions in the context of active immunotherapyis relevant to the raising antibodies in experimental animals forpassive immunotherapy and for the generation of diagnostic methods.

6.1 Active Immunotherapy

According to the present invention, UspA1 or UspA2 polypeptides orUspA1- or UspA2-derived peptides, as discussed above, may be used asvaccine formulations to generate protective anti-M. catarrhalis antibodyresponses in vivo. By protective, it is only meant that the immunesystem of a treated individual is capable of generating a response thatreduces, to any extent, the clinical impact of the bacterial infection.This may range from a minimal decrease in bacterial burden to outrightprevention of infection. Ideally, the treated subject will not exhibitthe more serious clinical manifestations of M. catarrhalis infection.

Generally, immunoprophylaxis involves the administration, to a subjectat risk, of a vaccine composition. In the instant case, the vaccinecomposition will contains a UspA1 and/or UspA2 polypeptide orimmunogenic derivative thereof in a pharmaceutically acceptable carrier,diluent or excipient. As stated above, those of skill in the art areable, through a variety of mechanisms, to identify appropriate antigeniccharacteristics of UspA1 and UspA2 and, in so doing, develop vaccinesthat will achieve generation of immune responses against M. catarrhalis.

The stability and immunogenicity of UspA1 and UspA2 antigens may varyand, therefore, it may be desirable to couple the antigen to a carriermolecule. Exemplary carriers are KLH, BSA, human serum albumin,myoglobin, β-galactosidase, penicillinase, CRM₁₉₇ and bacterial toxoids,such as diphtheria toxoid and tetanus toxoid. Those of skill in the artare aware of proper methods by which peptides can be linked to carrierswithout destroying-their immunogenic value. Synthetic carriers such asmulti-poly-DL-alanyl-poly-L-lysine and poly-L-lysine also arecontemplated. Coupling generally is accomplished through amino orcarboxyl-terminal residues of the antigen, thereby affording the peptideor polypeptide the greatest chance of assuming a relatively “native”,conformation following coupling.

It is recognized that other protective agents could be coupled witheither a UspA1 or UspA2 antigen such that the UspA1 or UspA2 antigenacts as the carrier molecule. For example, agents which protect againstother pathogenic organisms, such as bacteria, viruses or parasites,could be coupled to either a UspA1 or UspA2 antigen to produce amultivalent vaccine or pharmaceutical composition which would be usefulfor the treatment or inhibition of both M. catarrhalis infection andother pathogenic infections. In particular, it is envisioned that eitherUspA1 or UspA2 proteins or peptides could serve as immunogenic carriersfor other vaccine components, for example, saccharides of pneumococcus,menigococcus or hemophylus influenza and could even be covalentlycoupled to these other components.

It also may be desirable to include in the composition any of a numberof different substances referred to as adjuvants, which are known tostimulate the appropriate portion of the immune system of the vaccinatedanimal. Suitable adjuvants for the vaccination of subjects (includingexperimental animals) include, but are not limited to oil emulsions suchas Freund's complete or incomplete adjuvant (not suitable for livestockuse), Marcol 52:Montanide 888 (Marcol is a Trademark of Esso, Montanideis a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65(containing peanut oil, mannide monooleate and aluminum monostearate),MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem ResearchInc., Hamilton, Utah), Stimulon™ (QS-21; Aquila Biopharmaceuticals Inc.,Wooster, Mass.), mineral gels such as aluminum hydroxide, aluminumphosphate, calcium phosphate and alum, surfactants such ashexadecylamine, octadecylamine, lysolecithin,dimethyl-dioctadecylammonium bromide,N,N-dioctadecyl-N,N′-bis(2-hydroxyethyl)-propanediamine,methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran,dextran sulfate, polyacrylic acid and carbopol, peptides and amino acidssuch as muramyl dipeptide, dimethylglycine, tuftsin and trehalosedimycolate. Agents include synthetic polymers of sugars (Carbopol),emulsion in physiologically acceptable oil vehicles such as mannidemono-oleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (Fluosol-DA) also may be employed.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: Solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

The vaccine preparations of the present invention also can beadministered following incorporation into non-toxic carriers such asliposomes or other microcarrier substances, or after conjugation topolysaccharides, proteins or polymers or in combination with Quil-A toform “iscoms” (immunostimulating complexes). These complexes can serveto reduce the toxicity of the antigen, delay its clearance from the hostand improve the immune response by acting as an adjuvant. Other suitableadjuvants for use this embodiment of the present invention include INF,IL-2, IL-4, IL-8, IL-12 and other immunostimulatory compounds. Furthersconjugates comprising the immunogen together with an integral membraneprotein of prokaryotic origin, such as TraT (see PCT/AU87/00107) mayprove advantageous.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The peptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescers, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

6.2 Passive Immunotherapy

Passive immunity is defined, for the purposes of this application, asthe transfer to an organism of an immune response effector that wasgenerated in another organism. The classic example of establishingpassive immunity is to transfer antibodies produced in one organism intoa second, immunologically compatible animal. By “immunologicallycompatible,” it is meant that the antibody can perform at least some ofits immune functions in the new host animal. More recently, as a betterunderstanding of cellular immune functions has evolved, it has becomepossible to accomplish passive immunity by transferring other effectors,such as certain kinds of lymphocytes, including cytotoxic and helper Tcells, NK cells and other immune effector cells. The present inventioncontemplates both of these approaches.

Antibodies, antisera and immune effector cells are raised using standardvaccination regimes in appropriate animals, as discussed above. Theprimary animal is vaccinated with at least a microbe preparation or onebacterial product or by-product according to the present invention, withor without an adjuvant, to generate an immune response. The immuneresponse may be monitored, for example, by measurement of the levels ofantibodies produced, using standard ELISA methods.

Once an adequate immune response has been generated, immune effectorcells can be collected on a regular basis, usually from blood draws. Theantibody fraction can be purified from the blood by standard means,e.g., by protein A or protein G chromatography. In an alternativepreferred embodiment, monoclonal antibody-producing hybridomas areprepared by standard means (Coligan et al., 1991). Monoclonal antibodiesare then prepared from the hybridoma cells by standard means. If theprimary hosts monoclonal antibodies are not compatible with the animalto be treated, it is possible that genetic engineering of the cells canbe employed to modify the antibody to be tolerated by the animal to betreated. In the human context, murine antibodies, for example, may be“humanized” in this fashion.

Antibodies, antisera or immune effector cells, prepared as set forthabove, are injected into hosts to provide passive immunity againstmicrobial infestation. For example, an antibody composition is preparedby mixing, preferably homogeneously mixing, at least one antibody withat least one pharmaceutically or veterinarally acceptable carrier,diluent, or excipient using standard methods of pharmaceutical orveterinary preparation. The amount of antibody required to produce asingle dosage form will vary depending upon the microbial species beingvaccinated against, the individual to be treated and the particular modeof administration. The specific dose level for any particular individualwill depend upon a variety of factors including the age, body weight,general health, sex, and diet of the individual, time of administration,route of administration, rate of excretion, drug combination and theseverity of the microbial infestation.

The antibody composition may be administered intravenously,subcutaneously, intranasally, orally, intramuscularly, vaginally,rectally, topically or via any other desired route. Repeated dosings maybe necessary and will vary, for example, depending on the clinicalsetting, the particular microbe, the condition of the patient and theuse of other therapies.

6.3 DNA Immunization HC

The invention also relates to a vaccine comprising a nucleic acidmolecule encoding a UspA1, UspA2 protein or a peptide comprising SEQ IDNO:17 wherein said UspA1, UspA2 protein or peptide retainsimmunogenicity and, when incorporated into an immunogenic composition orvaccine and administered to a vertebrate, provides protection withoutinducing enhanced disease upon subsequent infection of the vertebratewith M. catarrhalis, and a physiologically acceptable vehicle. Such avaccine is referred to herein as a nucleic acid vaccine or DNA vaccineand is useful for the genetic immunization of vertebrates.

The term, “genetic immunization”, as used herein, refers to inoculationof a vertebrate, particularly a mammal such as a mouse or human, with anucleic acid vaccine directed against a pathogenic agent, particularlyM. catarrhalis, resulting in protection of the vertebrate against M.catarrhalis. A “nucleic acid vaccine” or “DNA vaccine” as used herein,is a nucleic acid construct comprising a nucleic acid molecule encodingUspA1, UspA2 or an immunogenic epitope comprising SEQ ID NO:17. Thenucleic acid construct can also include transcriptional promoterelements, enhancer elements, splicing signals, termination andpolyadenylation signals, and other nucleic acid sequences.

The nucleic acid vaccine can be produced by standard methods. Forexample, using known methods, a nucleic acid (e.g., DNA) encoding UspA1or UspA2 can be inserted into an expression vector to construct anucleic acid vaccine (see Maniatis et al., 1989). The individualvertebrate is inoculated with the nucleic acid vaccine (i.e., thenucleic acid vaccine is administered), using standard methods. Thevertebrate can be inoculated subcutaneously, intravenously,intraperitoneally, intradermally, intramuscularly, topically, orally,rectally, nasally, buccally, vaginally, by inhalation spray, or via animplanted reservoir in dosage formulations containing conventionalnon-toxic, physiologically acceptable carriers or vehicles.Alternatively, the vertebrate is inoculated with the nucleic acidvaccine through the use of a particle acceleration instrument (a “genegun”). The form in which it is administered (e.g., capsule, tablet,solution, emulsion) will depend in part on the route by which it isadministered. For example, for mucosal administration, nose drops,inhalants or suppositories can be used.

The nucleic acid vaccine can be administered in conjunction with anysuitable adjuvant. The adjuvant is administered in a sufficient amount,which is that amount that is sufficient to generate an enhanced immuneresponse to the nucleic acid vaccine. The adjuvant can be administeredprior to (e.g., 1 or more days before) inoculation with the nucleic acidvaccine; concurrently with (e.g., within 24 hours of) inoculation withthe nucleic acid vaccine; contemporaneously (simultaneously) with thenucleic acid vaccine (e.g., the adjuvant is mixed with the nucleic acidvaccine, and the mixture is administered to the vertebrate); or after(e.g. 1 or more days after) inoculation with the nucleic acid vaccine.The adjuvant can also be administered at more than one time (e.g., priorto inoculation with the nucleic acid vaccine and also after inoculationwith the nucleic acid vaccine). As used herein, the term “in conjunctionwith” encompasses any time period, including those specificallydescribed herein and combinations of the time periods specificallydescribed herein, during which the adjuvant can be administered so as togenerate an enhanced immune response to the nucleic acid vaccine (e.g.,an increased antibody titer to the antigen encoded by the nucleic acidvaccine, or an increased antibody titer to M. catarrhalis). The adjuvantand the nucleic acid vaccine can be administered at approximately thesame location on the vertebrate; for example, both the adjuvant and thenucleic acid vaccine are administered at a marked site on a limb of thevertebrate.

In a particular embodiment, the nucleic acid construct isco-administered with a transfection-facilitating agent. In a preferredembodiment, the transfection-facilitating agent is dioctylglycylspermine(DOGS) (as exemplified in published PCT application publication no. WO96/21356 and incorporated herein by reference). In another embodiment,the transfection-facilitating agent is bupivicaine (as exemplified inU.S. Pat. No. 5,593,972 and incorporated herein by reference).

6.4 Animal Model for Testing Efficacy of Therapies

The evaluation of the functional significance of antibodies to surfaceantigens of M. catarrhalis has been hampered by the lack of a suitableanimal model. The relative lack of virulence of this organism foranimals rendered identification of an appropriate model system difficult(Doern, 1986). Attempts to use rodents, including chinchillas, to studymiddle ear infections caused by M. catarrhalis were unsuccessful, likelybecause this organism cannot grow or survive in the middle ear of thesehosts (Doyle, 1989).

Murine short-term pulmonary clearance models have now been developed(Unhanand et al., 1992; Verghese et al., 1990) which permit anevaluation of the interaction of M. catarrhalis with the lowerrespiratory tract as well as assessment of pathologic changes in thelungs. This model reproducibly delivers an inoculum of bacteria to alocalized peripheral segment of the murine lung. Bacteria multiplywithin the lung, but are eventually cleared as a result of (i) residentdefense mechanisms, (ii) the development of an inflammatory response,and/or (iii) the development of a specific immune response. Using thismodel, it has been demonstrated that serum IgG antibody can enter thealveolar spaces in the absence of an inflammatory response and enhancepulmonary clearance of nontypable H. influenzae (McGehee et al., 1989),a pathogen with a host range and disease spectrum nearly identical tothose of M. catarrhalis.

7.0 SCREENING ASSAYS

In still further embodiments, the present invention provides methods foridentifying new M. catarrhalis inhibitory compounds, which may be termedas “candidate substances,” by screening for immunogenic activity withpeptides that include one or more mutations to the identifiedimmunogenic epitopic region. It is contemplated that such screeningtechniques will prove useful in the general identification of anycompound that will serve the purpose of inhibiting, or even killing, M.catarrhalis, and in preferred embodiments, will provide candidatevaccine compounds.

It is further contemplated that useful compounds in this regard will inno way be limited to proteinaceous or peptidyl compounds. In fact, itmay prove to be the case that the most useful pharmacological compoundsfor identification through application of the screening assays will benon-peptidyl in nature and, e.g., which will serve to inhibit bacterialprotein transcription through a tight binding or other chemicalinteraction. Candidate substances may be obtained from libraries ofsynthetic chemicals, or from natural samples, such as rain forest andmarine samples.

To identify a M. catarrhalis inhibitor, one would simply conductparallel or otherwise comparatively controlled immunoassays and identifya compound that inhibits the phenotype of M. catarrhalis. Those of skillin the art are familiar with the use of immunoassays for competitivescreenings (for example refer to Sambrook et al. 1989).

Once a candidate substance is identified, one would measure the abilityof the candidate substance to inhibit M. catarrhalis in the presence ofthe candidate substance. In general, one will desire to measure orotherwise determine the activity of M. catarrhalis in the absence of theadded candidate substance relative to the activity in the presence ofthe candidate substance in order to assess the relative inhibitorycapability of the candidate substance.

7.1 Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art as will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, and subjected to DNA polymerizingenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene using sitedirected mutagenesis is provided as a means of producing potentiallyuseful species and is not meant to be limiting, as there are other waysin which sequence variants of genes may be obtained. For example,recombinant vectors encoding the desired gene may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

7.2 Second Generation Inhibitors

In addition to the inhibitory compounds initially identified, theinventor also contemplates that other sterically similar compounds maybe formulated to mimic the key portions of the structure of theinhibitors. Such compounds, which may include peptidomimetics of peptideinhibitors, may be used in the same manner as the initial inhibitors.

Certain mimetics that mimic elements of protein secondary structure aredesigned using the rationale that the peptide backbone of proteinsexists chiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions. A peptide mimetic is thus designed topermit molecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

The generation of further structural equivalents or mimetics may beachieved by the techniques of modeling and chemical design known tothose of skilled in the art. The art of computer-based chemical modelingis now well known. Using such methods, a chemical that specificallyinhibits viral transcription elongation can be designed, and thensynthesized, following the initial identification of a compound thatinhibits RNA elongation, but that is not specific or sufficientlyspecific to inhibit viral RNA elongation in preference to human RNAelongation. It will be understood that all such sterically similarconstructs and second generation molecules fall within the scope of thepresent invention.

8.0 DIAGNOSING M. CATARRHALIS INFECTIONS

8.1 Amplification and PCR™

Nucleic acid sequence used as a template for amplification is isolatedfrom cells contained in the biological sample, according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a cDNA.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to UspA1 or UspA2 protein or a mutant thereof arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and each incorporated herein by reference inentirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g. Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ (RT-PCR™) amplification procedure may beperformed in order to quantify the amount of mRNA amplified or toprepare cDNA from the desired mRNA. Methods of reverse transcribing RNAinto cDNA are well known and described in Sambrook et al., 1989.Alternative methods for reverse transcription utilize thermostable,RNA-dependent DNA polymerases. These methods are described in WO90/07641, filed Dec. 21, 1990, incorporated herein by reference.Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., PCT Application WO88/10315, incorporated herein by reference. In NASBA, the nucleic acidscan be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/0700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, 1990, incorporated by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

All the essential materials and reagents required for detecting P-TEFbor kinase protein markers in a biological sample may be assembledtogether in a kit. This generally will comprise preselected primers forspecific markers. Also included may be enzymes suitable for amplifyingnucleic acids including various polymerases (RT, Taq, etc.),deoxynucleotides and buffers to provide the necessary reaction mixturefor amplification.

Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachmarker primer pair. Preferred pairs of primers for amplifying nucleicacids are selected to amplify the sequences specified in SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ IDNO:12 or SEQ ID NO:14 or SEQ ID NO:16 such that, for example, nucleicacid fragments are prepared that include a contiguous stretch ofnucleotides identical to for example about 15, 20, 25, 30, 35, etc.; 48,49, 50, 51, etc.; 75, 76, 77, 78, 79, 80 etc.; 100, 101, 102, 103 etc.;118, 119, 120, 121 etc.; 127, 128, 129, 130, 131, etc.; 316, 317, 318,319, etc.; 322, 323, 324, 325, 326, etc.; 361, 362, 363, 364, etc.; 372,373, 374, 375, etc. of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16,so long as the selected contiguous stretches are from spatially distinctregions. Similar fragments may be prepared which are identical orcomplimentary to, for example, SEQ ID NO:1 such that the fragments donot hybridize to, for example, SEQ ID NO:3.

In another embodiment, such kits will comprise hybridization probesspecific for UspA1 or UspA2 proteins chosen from a group includingnucleic acids corresponding to the sequences specified in SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ IDNO:12 or SEQ ID NO:14 or SEQ ID NO:16 or to intermediate lengths of thesequences specified. Such kits generally will comprise, in suitablemeans, distinct containers for each individual reagent and enzyme aswell as for each marker hybridization probe.

8.2 Other Assays

Other methods for genetic screening to accurately detect M. catarrhalisinfections that alter normal cellular production and processing, ingenomic DNA, cDNA or RNA samples may be employed, depending on thespecific situation.

For example, one method of screening for genetic variation is based onRNase cleavage of base pair mismatches in RNA/DNA and RNA/RNAheteroduplexes. As used herein, the term “mismatch” is defined as aregion of one or more unpaired or mispaired nucleotides in adouble-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definitionthus includes mismatches due to insertion/deletion mutations, as well assingle and multiple base point mutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. After the RNase cleavage reaction, the RNase is inactivated byproteolytic digestion and organic extraction, and the cleavage productsare denatured by heating and analyzed by electrophoresis on denaturingpolyacrylamide gels. For the detection of mismatches, thesingle-stranded products of the RNase A treatment, electrophoreticallyseparated according to size, are compared to similarly treated controlduplexes. Samples containing smaller fragments (cleavage products) notseen in the control duplex are scored as +.

Currently available RNase mismatch cleavage assays, including thoseperformed according to U.S. Pat. No. 4,946,773, require the use ofradiolabeled RNA probes. Myers and Maniatis in U.S. Pat. No. 4,946,773describe the detection of base pair mismatches using RNase A. Otherinvestigators have described the use of E. coli enzyme, RNase I, inmismatch assays. Because it has broader cleavage specificity than RNaseA, RNase I would be a desirable enzyme to employ in the detection ofbase pair mismatches if components can be found to decrease the extentof non-specific cleavage and increase the frequency of cleavage ofmismatches. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is shown in their literature to cleave three out of four knownmismatches, provided the enzyme level is sufficiently high.

The RNase protection assay was first used to detect and map the ends ofspecific mRNA targets in solution. The assay relies on being able toeasily generate high specific activity radiolabeled RNA probescomplementary to the mRNA of interest by in vitro transcription.Originally, the templates for in vitro transcription were recombinantplasmids containing bacteriophage promoters. The probes are mixed withtotal cellular RNA samples to permit hybridization to theircomplementary targets, then the mixture is treated with RNase to degradeexcess unhybridized probe. Also, as originally intended, the RNase usedis specific for single-stranded RNA, so that hybridized double-strandedprobe is protected from degradation. After inactivation and removal ofthe RNase, the protected probe (which is proportional in amount to theamount of target mRNA that was present) is recovered and analyzed on apolyacrylamide gel.

The RNase Protection assay was adapted for detection of single basemutations. In this type of RNase A mismatch cleavage assay, radiolabeledRNA probes transcribed in vitro from wild type sequences, are hybridizedto complementary target regions derived from test samples. The testtarget generally comprises DNA (either genomic DNA or DNA amplified bycloning in plasmids or by PCR™, although RNA targets (endogenous, mRNA)have occasionally been used. If single nucleotide (or greater) sequencedifferences occur between the hybridized probe and target, the resultingdisruption in Watson-Crick hydrogen bonding at that position(“mismatch”) can be recognized and cleaved in some cases bysingle-strand specific ribonuclease. To date, RNase A has been usedalmost exclusively for cleavage of single-base mismatches, althoughRNase I has recently been shown as useful also for mismatch cleavage.There are recent descriptions of using the MutS protein and otherDNA-repair enzymes for detection of single-base mismatches.

9.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example I Sequence Analysis and Characterization of uspA1

Bacterial strains and culture conditions. M. catarrhalis strains 035E,046E, TTA24, 012E, FR2682, and B21 have been previously described(Helminen et al., 1993a; Helminen et al., 1994; Unhanand et al., 1992).M. catarrhalis strains FR3227 and FR2336 were obtained from RichardWallace, University of Texas Health Center, Tyler, Tex. M. catarrhalisstrain B6 was obtained from Elliot Juni, University of Michigan, AnnArbor, Mich. M. catarrhalis strain TTA1 was obtained from Steven Berk,East Tennessee State University, Johnson City, Tenn. M. catarrhalisstrain 25240 was obtained from the American Type Culture Collection,Rockville, Md. M. catarrhalis were routinely cultured in Brain HeartInfusion (BHI) broth (Difco. Laboratories, Detroit, Mich.) at 37° C. oron BHI agar plates in an atmosphere of 95% air-5% CO₂ . Escherichia colistrains LE392 and XL1-Blue MRF′ (Stratagene, La Jolla, Calif.) weregrown on Lubria-Bertani medium (Maniatis et al., 1982) supplemented withmaltose (0.2% w/v) and 10 mM MgSO₄ at 37° C., with antimicrobialsupplementation as necessary.

Monoclonal antibodies (MAbs). MAb 17C7 is a murine IgG antibody reactivewith the UspA proteinaceous material of all M. catarrhalis strainstested to date (Helminen et al., 1994). Additional MAbs specific forUspA material (i.e., 16A7, 17B1, and 5C12) were produced for this studyby fusing spleen cells from mice immunized with outer membrane vesiclesfrom M. catarrhalis 035E with the SP2/0-Ag14 plasmacytoma cell line, asdescribed (Helminen et al., 1993a). These MAbs were used in the form ofhybridoma culture supernatant fluid in western blot and dot blotanalyses.

Cloning vectors. Plasmid and bacteriophage cloning vectors utilized inthis work and the recombinant derivatives of these vectors are listed inTable VI.

TABLE VI Bacteriophages And Plasmids Bacteriophage or plasmidDescription Source Bacteriophage LambdaGEM-11 Cloning vector PromegaCorp. (Madison, WI) MEH200 LambdaGEM-11 containing an (Helminen et al.,11 kb insert of M. catarrhalis 1994) strain 035E DNA encoding the UspAproteinaceous material ZAP Express Cloning vector Stratagene USP100 ZAPExpress with a 2.7 kb This study fragment of DNA (containing the uspA1)amplified from the chromosome of M. catarrhalis strain 035E PlasmidspBluescript II SK+ Cloning vector, Amp^(R) Stratagene (pBS) pJL501.6 pBScontaining the 1.6 kb This study BglII-EcoRI fragment from MEH200pJL500.5 pBS containing the 600-bp BglII This study fragment from MEH200MEH200, the original recombinant bacteriophage clone that producedplaques reactive with the UspA-specific MAb 17C7, has been describedpreviously (Helminen et al., 1994).

Genetic techniques. Standard recombinant DNA techniques includingplasmid isolation, restriction enzyme digestions, DNA modifications,ligation reactions and transformation of E. coli are familiar to thoseof skill in the art and were performed as previously described (Maniatiset al., 1982; Sambrook et al., 1989).

Polymerase Chain Reaction (PCR™). PCR™ was performed using the GeneAmpkit (Perkin-Elmer, Branchberg, N.J.). All reaction were carried outaccording to the manufacturer's instructions. To amplify products fromtotal genomic DNA, 1 μg of M. catarrhalis chromosomal DNA and 100 ng ofeach primer were used in each 100 μl reaction.

Nucleotide sequence analysis. Nucleotide sequence analysis of DNAfragments in recombinant plasmids, in bacteriophage, or derived by PCR™was performed using an Applied Biosystems Model 373A automated DNAsequencer (Applied Biosystems, Foster City, Calif.) DNA sequenceinformation was analyzed using the Intelligenetics suite package andprograms from the University of Wisconsin Genetics Computer Groupsoftware analysis package (Devereux et al., 1984). Analysis of proteinhydrophilicity using the method of Kyte and Doolittle (1982) andanalysis of repeated amino acid sequences within the UspA protein wasperformed using the MacVector™ software protein matrix analysis package(Eastman Kodak Company, Rochester, N.Y.).

Identification of recombinant bacteriophage. Lysates were generated fromE. coli cells infected with recombinant bacteriophage by using the platelysis method as described (Helminen et al., 1994). MAb-based screeningof plaques formed by recombinant ZAP Express bacteriophage on E. coliXL1-Blue MRF′ cells was performed according to the manufacturer'sinstructions (Stratagene, La Jolla, Calif.). Briefly, nitrocellulosefilters soaked in 10 mM IPTG were applied to the surface of agar platesfive hours after bacteriophage infection of the bacterial lawn. Afterovernight incubation at 37° C., the nitrocellulose pads were removed,washed with PBS containing 0.5% (v/v) Tween 20 and 5% (w/v) skim milk(PBS-1) and incubated with hybridoma culture supernatant containing theMAb for 4 hours at room temperature. After four washes with PBS-T, PBS-Tcontaining ¹²⁵I-labeled goat anti-mouse IgG was applied to each pad.After overnight incubation at 4° C., the pads were washed four timeswith PBS-T, blotted dry, and exposed to film.

Characterization of M. catarrhalis protein antigens. Outer membranevesicles were prepared from BHI broth-grown M. catarrhalis cells by theEDTA-buffer method (Murphy and Loeb, 1989). Proteins present in thesevesicles were resolved by sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) using 7.5% (w/v) polyacrylamide separatinggels. These SDS-PAGE-resolved proteins were electrophoreticallytransferred to nitrocellulose and western blot analysis was performed asdescribed using MAb 17C7 as the primary antibody (Kimura et al., 1985).For western blot analysis of proteins encoded by DNA inserts inrecombinant bacteriophage, one part of a lysate frombacteriophage-infected E. coli cells was mixed with one partSDS-digestion buffer (Kimura et al., 1985) and this mixture wasincubated at 37° C. for 15 minutes prior to SDS-PAGE.

Features of the uspA1 gene and its encoded protein product. Thenucleotide sequence of the M. catarrhalis 035E uspA1 gene and thededuced amino acid sequence of the UspA1 protein are provided in SEQ IDNO:2 and SEQ ID NO:1, respectively. The open reading frame (ORF),containing 2,493 nucleotides, encoded a protein product of 831 aminoacids, with a calculated molecular mass of 88,271 daltons.

The predicted protein product of the uspA1 ORF had a pI or 4.7, washighly hydrophilic, and was characterized by extensively repeatedmotifs. The first motif consists of the consensus sequenceNXAXXYSXIGGGXN (SEQ ID NO:24), which is extensively repeated betweenamino acid residues 80 and 170. The second region, from amino acidresidues 320 to 460, contains a long sequence which is repeated threetimes in its entirety, but which also contains smaller units which arerepeated several times themselves. This “repeat within a repeat”arrangement is also true of the third region, which extends from aminoacid residues 460 to 600. This last motif consists of many repeats ofthe small motif QADI (SEQ ID NO:25) and two large repeats which containthe QADI (SEQ ID NO:25) motif within themselves.

Similarity of UspA1 to other proteins. A BLAST-X search (Altschul etal., 1990; Gish and States, 1993) of the available databases forproteins with significant homology to UspA1 indicated that theprokaryotic proteins that were most similar to this M. catarrhalisantigen were a putative adhesin of H. influenzae Rd (GenBank accessionnumber U32792) (Fleischmann et al., 1995), the Hia adhesin fromnontypable H. influenzae (GenBank accession number U38617) (Barenkampand St. Geme III, 1996), and the YadA invasin of Yersinia enterocolitica(Skurnik and Wolf-Watz, 1989) (SwissProt:P31489). When the GAP alignmentprogram (Devereux et al., 1984) was used to compare the UspA1 sequenceto that of these and closely related bacterial adhesins, UspA1 proved tobe 25% identical and 47% similar to the E. coli AIDA-I adhesin fromenteropathogenic E. coli (Benz and Schmidt, 1989; Benz and Schmidt,1992b), 23% identical and 46% similar to Hia (Barenkamp and St. GemeIII, 1996), and 24% identical and 43% similar to YadA (Skurnik andWolf-Watz, 1989). Other proteins retrieved from database searches ashaving homology with UspA1 included myosin heavy chains from a number ofspecies.

Example II Two Genes Encode the Proteins UspA1 and UspA2

MAb 17C7 binds to a very high molecular weight proteinaceous material ofM. catarrhalis, designated UspA, that migrates with an apparentmolecular weight (in SDS-PAGE) of at least 250 kDa. This same MAb alsoreacts with another antigen band of approximately 100 kDa, as describedin U.S. Pat. No. 5,552,146 and incorporated herein by reference, and itis bound by a phage lysate from E. coli infected by a recombinantbacteriophage that contained a fragment of M. catarrhalis chromosomalDNA. The M. catarrhalis proteinaceous material in the phage lysate thatbinds this MAb migrates at a rate similar or indistinguishable from thatof the native UspA material (Helminen et al., 1994).

Analysis of uspA1. Nucleotide sequence analysis of the M. catarrhalisstrain O35E gene expressed by the recombinant bacteriophage, designateduspA1, revealed the presence of an ORF encoding a predicted proteinproduct with a molecular mass of 88,271 (SEQ ID NO:1). The use of theuspA1 ORF in an in vitro DNA-directed protein expression system revealedthat the protein encoded by the uspA1 gene migrated in SDS-PAGE with anapparent molecular weight of about 120 kDa. (Those of skill in the artwill be aware that denaturing processes, such as SDS-PAGE, can alter themigration rate of proteins such that the apparent molecular weight ofthe denatured protein is somewhat different than the predicted molecularweight of the non-denatured protein.) In addition, when the uspA1 ORFwas introduced into a bacteriophage vector, the recombinant E. colistrain containing this recombinant phage expressed a protein thatmigrated in SDS-PAGE apparently at the same rate as the native UspAprotein from M. catarrhalis.

Southern blot analysis of chromosomal DNA from several M. catarrhalisstrains, using a 0.6 kb BglII-PvuII fragment derived from the cloneduspA1 gene as the probe, revealed that, with several strains, there weretwo distinct restriction fragments that bound this uspA1-derived probe(FIG. 1), indicating that M. catarrhalis possessed a second gene hadsome similarity to the uspA1 gene.

Native very high molecular weight UspA proteinaceous material from M.catarrhalis strain O35E was resolved by SDS PAGE, electroeluted, anddigested with a protease. N-terminal acid sequence analysis of some ofthe resultant peptides revealed that the amino acid sequences of severalpeptides did not match that of the deduced amino acid sequence of UspA1.Other peptides obtained from this experiment were similar to thosepresent in the deduced amino acid sequence but not identical.

Protease and cyanogen bromide (CNBr) Cleavage of High Molecular WeightUspA Proteinaceous Material: Three tenths (0.3) mg of purified very highmolecular weight UspA proteinaceous material (at the time of thepurification this material was thought to be a single protein) wasprecipitated with 90% ethanol and the pellet was resuspended in 100 mlof 88% formic acid containing 12M urea. Following resuspension, 100 mlof 88% formic acid containing 2M CNBr was added and the mixture wasincubated in the dark overnight at room temperature. One ml (2.0 mg) ofpurified UspA material was added directly to a vial containing 25 mg ofeither trypsin or chymotrypsin. The reaction mixtures were incubated for˜48 hours. at 37° C. One ml (2.0 mg) of purified UspA material was addeddirectly to a vial containing 15 mg of endoproteinase Lys-C. Thereaction mixtures were incubated for about 48 hours at 37° C.

The cleavage reaction mixtures were clarified by centrifugation in anEppendorf™ centrifuge at 12,000 rpm for 5 minutes. The clarifiedsupernatant was loaded directly onto a Vydac C4 HPLC column using amobile phase of 0.1% (v/v) aqueous trifluoroacetic acid (Solvent A) andacetonitrile:H₂O:trifluoroacetic acid, 80:20:0.1 (v/v/v) (Solvent B) ata flow rate of 1.0 ml/min. The reaction mixtures were washed onto thecolumn with 100% Solvent A followed by elution of cleavage fragmentsusing a 30 minutes linear gradient (0-100%) of Solvent B. Fractions werecollected manually, dried overnight in a Speed-Vac and resuspended inHouse Pure Water. The resuspended HPLC-separated fractions weresubjected to SDS-PAGE analysis using 10-18% gradient gels in aTris-Tricine buffer system. The fractions which exhibited a singlepeptide band were submitted for direct N-terminal sequence analysis.Fractions displaying multiple peptide bands were transferred fromSDS-PAGE onto a PVDF membrane and individual bands excised and submittedfor N-terminal sequence analysis.

The N-terminal amino acid sequences of these fragments then weredetermined using an Applied Biosystems Model 477A PTH Analyzer (AppliedBiosystems, Foster City, Calif., U.S.A.). A summary of these sequencesis given in Table VII. About half of the sequences were found to matchthe sequence deduced from the uspA1 gene, while the other half did not.Attempts at shifting the reading frame of the uspA1 gene sequence failedto account for the non-matching peptide sequences, indicating that thehigh molecular weight UspA protein may comprise either a multimer ofmore than one distinct protein or distinct multimers of two differentproteins.

TABLE VII Summary of the N-terminal Sequences of Internal PeptideFragments Digest Sequence^(a) CNBr AAQAALSGLFVPYSVGKFNATAALGGYGSK SEQ IDNO:26 GKITKNAARQENG SEQ ID NO:27 LysC Digest #1 VIGDLGRKV SEQ ID NO:28ALEXNVEEGL SEQ ID NO:29 ALESNVEEGLXXLS SEQ ID NO:30 ALEFNGE SEQ ID NO:31LysC Digest #2 SITDLGXKV SEQ ID NO:32 SITDLGTIVDGFXXX SEQ ID NO:33SITDLGTIVD SEQ ID NO:34 Trypsin VDALXTKVNALDXKVNSDXT SEQ ID NO:35LLAEQQLNGKTLTPV SEQ ID NO:36 AKHDAASTEKGKMD SEQ ID NO:37 ALESNVEEGLLDLSGSEQ ID NO:38 Trypsin Digest #1 NQNTLIEKTANK SEQ ID NO:39 IDKNEYSIK SEQID NO:40 SITDLGTK SEQ ID NO:41 Trypsin Digest #2 NQNTLIEK SEQ ID NO:42ALHEQQLETLTK SEQ ID NO:43 NSSD SEQ ID NO:44 NKADADASFETLTK SEQ ID NO:45FAATAIAKDK SEQ ID NO:46 KASSENTQNIAK SEQ ID NO:47 RLLDQK SEQ ID NO:48Chymotrypsin AATADAITKNGX SEQ ID NO:49 AKAXAANXDR SEQ ID NO:50 Digest ofresearch NQADIAQNQTDIQDLAAYNELQ grade UspA with SEQ ID NO:51cys-C-endopeptidase NQADIANNINNIYELAQQQDQ SEQ ID NO:52 YNERQTEAIDALN SEQID NO:53 ILGDTAIVSNSQD SEQ ID NO:54 ^(a)Certain residues of severalpeptides could not be verified and these ambiguities are shown by an“X” in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:49 and SEQ ID NO:50. In SEQ ID NO:29 the ambiguousresidue is likely to be a serine; in SEQ ID NQ:33, position 13 is likelyto be aspartic acid, position 14 is likely to be glycine and position 15is likely to be arginine; in SEQ II) NQ:35 both positions 13 and 19 arelikely to be serines; in SEQ ID NO:49 the ambiguous residue is likely tobe an asparagine; and in SEQ ID NO:50 position 4 is likely to be serineand position 8 is likely to be threonine.

Additional attempts to resolve the very high molecular weight UspAprotein band from M. catarrhalis strain O35E by SDS-PAGE, followed byelectroelution and digestion with proteases or with cyanogen bromide,again yielded a number of peptides which were sequenced. Severalpeptides (peptides 1-6, Table VIII) were obtained. The amino acidsequence of which was identical or very similar to that deduced from thenucleotide sequence of the uspA1 gene. However, several additionalpeptides, peptides 7-10, Table VIII, were not present in the deducedamino acid sequence. This finding substantiated the suggestion that asecond protein was present in the UspA antigen preparation.

TABLE VIII Peptide # Amino acid sequence Matching or closely matchingpeptides: Peptide 1 KALESNVEEGLLDLSGR (SEQ ID NO:55) Peptide 2ALESNVEEGLLELSGRTIDQR (SEQ ID NO:56) Peptide 3 NQAHIANNINXIYELAQQQDQK(SEQ ID NO:57) Peptide 4 NQADIAQNQTDIQDLAAYNELQ (SEQ ID NO:58) Peptide 5ATHDYNERQTEA (SEQ ID NO:59) Peptide 6 KASSENTQNIAK (SEQ ID NO:60)Nonmatching peptides: Peptide 7 MILGDTAIVSNSQDNKTQLKFYK (SEQ ID NO:61)Peptide 8 AGDTIIPLDDDXXP (SEQ ID NO:62) Peptide 9 LLHEQQLXGK (SEQ IDNO:63) Peptide 10 IFFNXG (SEQ ID NO:64) ^(a)Certain residues of severalpeptides could not be verified and tbese ambiguities are shown by an“X” in SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:64.

Further evidence corroborating the assertion that the high molecularweight UspA proteinaceous material was either a multimer of more thanone distinct protein or distinct multimers of two different proteins wasderived from earlier electrospray mass spectroscopic analysis whichpredicted that a monomer of the UspA material had a molecular weight of59,500. This approximately 60 kDa protein reacted immunogenically withthe MAbs 17C7, 45-2, 13-1, and 29-31, in contrast to the UspA1 proteinwhich only cross-reacted with MAb 17C7. The fact that MAb 17C7 reactedwith both isolated proteins suggested that this Mab recognized anepitope common to both proteins.

Preparation of mutant uspA1 construct. The nucleotide sequence of thecloned uspA1 gene was used to construct an isogenic uspA1 mutant.Oligonucleotide primers (BamHI-ended P1 and P16 in Table IX) were usedto amplify a truncated version of the usAp1 ORF from M. catarrhalisstrain O35E chromosomal DNA; this PCR™ product was cloned into the BamHIsite of the plasmid vector pBluescript II SK+. A 0-6 kb BglII fragmentfrom the middle of this cloned fragment was excised and was replaced bya BamHI-ended cassette encoding kanamycin resistance. This new plasmidwas grown in E. coli DH5α, purified by column chromatography, linearizedby digestion with EcoRI, precipitated, and then dissolved in water. Thislinear DNA molecule was used to electroporate the wild-type M.catarrhalis strain O35E, using a technique described previously(Helminen et al., 1993b). Approximately 5,000 kanamycin-resistanttransformants were obtained; several picked at random were found to bestill reactive with MAb 17C7. One of these kanamycin-resistant cloneswas randomly chosen for further examination and Southern blot analysisconfirmed that this mutant was isogenic.

Analysis of products expressed by the uspA1 mutant. When whole celllysates of both the wild-type M. catarrhalis strain and this mutant weresubjected to SDS-PAGE, both the wild-type strain and the mutant strainstill expressed the very high-molecular-weight band originallydesignated as UspA. However, a protein of approximately 120 kDa wasfound to be missing in the mutant strain (FIG. 2A). The fact that boththis mutant and the wild-type parent strain still expressed a very highmolecular weight antigen reactive with MAb 17C7 (FIG. 2B) indicated thatthere had to be a second gene in M. catarrhalis strain O35E that encodeda MAb 17C7-reactive antigen. Furthermore, it should be noted thatEDTA-extracted outer membrane vesicles of both the wild-type strain(FIG. 2C, lanes 5 and 7) and mutant strain (FIG. 2C, lanes 6 and 8)possessed a protein of approximately 70-80 kDa that was reactive withMAb 17C7. This approximately 70-80 kDa band likely represents one form,perhaps the monomeric form, of the product of a second gene encoding theMAb 17C7-reactive epitope.

It is important to note that, when chromosomal DNA from both thewild-type parent strain and the mutant were digested with PvuII andprobed in Southern blot analysis with a 0.6 kb BglII-PvuII fragmentderived from the uspA1 gene, the wild-type strain exhibited a 2.6 kbband and a 2.8 kb band which bound this probe (FIG. 3). In contrast, themutant strain had a 2.6 kb band and a 3.4 kb band that bound this probe.The presence of the 3.4 kb band was the result of the insertion of thekan cartridge into the deletion site in the uspA1 gene.

Example III Characterization of UspA2 and uspA2

Construction of fusion proteins. The epitope which binds MAb 17C7 waslocalized by using the nucleotide sequence of the uspA1 gene describedabove to construct fusion proteins. First, fusion proteins containingfive peptides spanning the UspA1 protein were constructed by using thepGEX4T-2 protein fusion system Pharmacia LKB). The oligonucleotideprimers used in PCR™ to amplify the desired nucleotide sequences from M.catarrhalis stain O35E chromosomal DNA are listed in Table IX. Each ofthese had either a BamHI site or a XhoI site at the 5′ end, therebyallowing directional in-frame cloning of the amplified product into theBamHI- and XhoI-digested vector. When recombinant E. coli strainsexpressing each of these five fusion proteins were used in a colony blotradioimmunoassay, only fusion protein MF-4 readily bound MAb 17C7.Further analysis of the uspA1-derived nucleotide sequence in the MF-4fusion construct involved the production of fusion proteins containing79 amino acid residues (MF-4-1) and 123 amino acid residues (MF-4-2)derived from the MF-4 fusion protein (Table IX). These two fusionproteins both bound MAb 17C7 (Table IX). FIG. 4 depicts the western blotreactivity of MAb 17C7 with the MF-4-1 fusion protein. These two fusionproteins had in common only a 23-residue region NNINNIYELAQQQDQHSSDIKTL(SEQ ID NO:65), suggesting that this 23-residue region, designated asthe “3Q” peptide, contains the epitope that binds MAb 17C7.

TABLE IX PCR™ primers used for the production of usp A1 gene fragmentsfor use in the construction of fusion proteins and mutagenesis and thereactivity of the resulting fusion protein with MAb 17C7 Reactivity withFragment Generated: Primer Pair^(a) MAb 17C7 MF-3 P5-P8 − MF-4 P6-P13 +MF-4.1 P7-P12 + MF-42 P11-P13 + ^(a)primer sequences are as follows: P5GGTGCAGGTCAGATCAGTGAC SEQ ID NO:66 P6 GCCACCAACCAAGCTGAC SEQ ID NO:67 P7AGCGGTCGCCTGCTTGATCAG SEQ ID NO:68 P8 CTGATCAAGCAGGCGACCGCT SEQ ID NO:69P11 CAAGATCTGGCCGCTTACAA SEQ ID NO:70 P12 TTGTAAGCGGCCAGATCTTG SEQ IDNO:71 P13 TGCATGAGCCGCAAACCC SEQ ID NO:72

Elucidation of the MAb 17C7 Epitope. It is important to note that thenucleotide sequence encoding this 23-residue polypeptide (i.e., the 3Qpeptide) was present in the 0.6 kb BglII-PvuII fragment used in theSouthern blot analysis described in Example II. This finding suggestedthat the epitope that bound MAb 17C7 might be encoded by DNA present inboth the 2.6 and 2.8 kb PvuII fragments, with the 2.8 kb PvuII fragmentbeing derived from the cloned uspA1 gene and the 2.6 kb PvuII fragmentrepresenting all or part of another gene encoding this same epitope.

A ligation-based PCR™ system was used to verify this finding.Chromosomal DNA from the mutant strain was digested to completion withPvuII and was resolved by agarose gel electrophoresis. Fragments rangingin size from 2-3 kb were excised from the agarose, blunt-ended, andligated into the EcoRV site in pBluescript II SK+ This ligation reactionmixture was precipitated and used in a PCR™ amplification reaction. EachPCR™ reaction contained either the T3 or T7 primer derived from the DNAencoding the 3Q peptide. This approach yielded a 1.7 kb product with theT3 and P10 primers and a 0.9 kb product from the T7 and P9 primers (FIG.5). The sum of these two bands is the same as the 2.6 kb size of thedesired DNA fragment.

Nucleotide sequence analysis of these two PCR™ products revealed twoincomplete ORFs which, when joined at the region encoding the 3Qpeptide, formed a 1,728-bp ORF encoding a protein with a calculatedmolecular weight of 62,483 daltons (SEQ ID NO:3). The amino acidsequence of this protein had 43% identity with that of UspA1. Closerexamination revealed that a region extending from amino acids 278-411 inthis second protein, designated UspA2, was nearly identical to theregion in UspA1 between amino acids 505538 (SEQ ID NO:1). Furthermore,these two regions both contain the 23-mer (the 3Q peptide) that likelycontains the epitope that binds MAb 17C7. It should also be noted thatthe four peptides from Table X (Peptides 7-10) that were not found inUspA1 were found to be identical or very similar to peptides in thededuced amino acid sequence of UspA2. In addition, the first sixpeptides listed in Table IX, which matched or were very similar topeptides in the deduced amino acid sequence of UspA1, also matchedpeptides found in the deduced amino acid sequence of UspA2.

Oligonucleotide primers P1 and P2 (Table IX) were used to amplify a2.5-2.6 kb fragment from M. catarrhalis strain O35E chromosomal DNA.Nucleotide sequence analysis of this PCR™ product was used to confirmthe nucleotide sequence of the uspA2 ORF determined from theligation-based PCR™ study. These results proved that M. catarrhalisstrain O35E contains two different ORFs (i.e., uspA1 and uspA2) whichencode the same peptide (i.e., the 3Q peptide) which likely binds MAb17C7. This 3Q peptide appears twice in UspA1 and once in UspA2 (SEQ IDNO:1 and SEQ ID NO:3).

The nucleotide sequences of the two DNA segments encoding these 3Qpeptides in uspA1 are nearly identical, with three nucleotides beingdifferent. These nucleotide differences did not cause a change in theamino acid sequence. The nucleotide sequence of the DNA segment encodingthe 3Q peptide in uspA2 is identical to the DNA encoding the first 3Qpeptide in UspA1.

As seen in FIG. 2C, lane 7, the three dominant MAb 17C7-reactive bandspresent in M. catarrhalis strain O35E outer membrane vesicles haveapparent molecular weights of greater than 200 kDa, approximately 120kDa, and approximately 70-80 kDa. It should be noted that the existenceof several MAb 17C7-reactive bands, with apparent molecular weights ofgreater than 200 kDa, approximately 120 kDa, and approximately 70-80 kDawas also apparent in U.S. Pat. No. 5,552,146 (FIG. 1, lane H).Therefore, the existence of at least more than one M. catarrhalisantigens reactive with MAb 17C7 was apparent as early as 1991. It is nowapparent that the approximately 120 kDa band likely represents themonomeric form of the UspA1 antigen and the approximately 70-80 kDa bandlikely represents the monomeric form of the UspA2 antigen from M.catarrhalis strain O35E. One or more than one of these species mayaggregate to form the very high molecular weight proteinaceous material(i.e. greater than 200 kDa) of the UspA antigen.

A new M. catarrhalis strain O35E genomic library was constructed in thebacteriophage vector ZAP Express (Stratagene, La Jolla, Calif.).Chromosomal DNA from this strain was partially digested with Sau3A1 and4-9 kb DNA fragments were ligated into the vector arms according to theinstructions obtained from the manufacturer. This library was amplifiedin E. coli MRF′. An aliquot of this library was diluted and plated andthe resultant plaques were screened for reactivity with MAb 17C7.Approximately 24 plaques which bound this MAb were detected; theresponsible recombinant bacteriophage were purified by the single plaqueisolation method, and the DNA insert from one of these bacteriophage wassubjected to nucleotide sequence analysis. Nucleotide sequence of the2.6 kb DNA fragment present in this recombinant bacteriophage revealedthat, on one end, it contained an incomplete ORF that encoded the 3Qpeptide. Until its truncation by the vector cloning site, the sequenceof this incomplete ORF was identical or nearly identical to that of theuspA2 ORF derived from the ligation-based PCR™ study describedimmediately above, providing further evidence that two genes which sharea common epitope encode the UspA antigen.

Example IV Purification of and Immunological Properties of the ProteinsUspA1 and UspA2

Materials and Methods

Bacteria. TTA24 and O35E isolates were as previously described inExample I. Additional isolates were obtained from the University ofRochester and the American Type Culture Collection (ATCC). The bacteriawere routinely passaged on Mueller-Hinton agar (Difco, Detroit, Mich.)incubated at 35° C. with 5% carbon dioxide. The bacteria used for thepurification of the protein were grown in sterile broth containing 10 gcasamino acids (Difco, Detroit, Mich.) and 15 g yeast extract (BBL,Cockeysville, N. Mex.) per liter. The isolates were stored at −70° C. inMueller-Hinton broth containing 40% glycerol.

Purification of UspA2. Bacterial cells (˜400 g wet wt. of M. catarrhalisO35E) were washed twice with 2 liters of pH 6.0, 0.03 M sodium phosphate(NaPO₄) containing 1.0% Triton® X-100 (TX-100) (J. T. Baker Inc.,Philipsburg, N.J.) (pH 6.0) by stirring at room temperature for 60 min.Cells containing the UspA2 protein were pelleted by centrifugation at13,700×g for 30 min at 4° C. Following-centrifugation, the pellet wasresuspended in 2 liters of pH 8.0, 0.03 MTris(hydroxymethyl)aminomethane-HCl (Tris-HCl) containing 1.0% TX-100and stirred overnight at 4° C. to extract the UspA2 protein. Cells werepelleted by centrifugation at 13,700×g for 30 nm in at 4° C. Thesupernatant, containing the UspA2 protein, was collected and furtherclarified by sequential microfiltration through a 0.8 μm membrane (CN.8,Nalge, Rochester, N.Y.) then a 0.45 μm membrane (cellulose acetate, lowprotein binding, Corning, Corning, N.Y.).

The entire filtered crude extract preparation was loaded onto a 50×217mm (˜200 ml) TMAE column [650(S), 0.025-0.4 mm, EM Separations,Gibbstown, N.J.] equilibrated with pH 8.0, 0.03 M Tris-HCl buffercontaining 0.1% TX-100 (THT). The column was washed with 400 ml ofequilibration buffer followed by 600 ml of 0.25 M NaCl in 0.03 M THT.UspA2 was subsequently eluted with 800 ml of 1.0 M NaCl in 0.03 M THT.Fractions were screened for UspA2 by SDS-PAGE and pooled. Pooledfractions (˜750 ml), containing UspA2, were concentrated approximatelytwo-fold by ultrafiltration using an Amicon stirred cell (Amicon Corp.,Beverly, Mass.) with a YM-100 membrane under nitrogen pressure. The TMAEconcentrate was split into two 175 ml aliquots and each aliquot bufferexchanged by passage over a 50×280 mm (˜550 ml) Sephadex G-25 (Coarse)column (Pharmacia Biotech, Piscataway, N.J.) equilibrated with pH 7.0,10 mM NaPO₄ containing 0.1% TX-100 (10 mM PT). The buffer exchangedmaterial was subsequently loaded onto a 50×217 mm (˜425 ml) ceramichydroxyapatite-column (Type I, 40 μm, Bio-Rad) equilibrated with 10 mMPT. The column was washed with 450 ml of the equilibration bufferfollowed by 900 ml of pH 7.0, 0.1M NaPO₄ containing 0.1% TX-100. UspA2was then eluted with a linear pH 7.0 NaPO₄ concentration gradientbetween 0.1 and 0.2 M NaPO₄ containing 0.1% TX-100. An additional volumeof pH 7.0, 0.2 M NaPO₄ containing 0.1% TX-100 was applied to the columnand collected to maximize the recovery of UspA2. Fractions were screenedfor UspA2 by SDS-PAGE and pooled. The column was then washed with 900 mlof pH 7.0, 0.5 M NaPO₄ containing 0.1% TX-100. The fractions from thiswash were screened for UspA1 by SDS-PAGE, pooled, and stored at 4° C.This pool was used for the purification of UspA1.

Purification of UspA-1. The UspA1 enriched fractions collected duringfour separate purifications of UspA2 were pooled. The combined UspA1pools were concentrated approximately threefold by ultrafiltration usingan Amicon stirred cell with a YM-100 membrane under nitrogen pressure.The UspA1 concentrate was split into two 175 ml aliquots and the bufferexchanged by passage over a 50×280 mm (˜550 ml) Sephadex G-25 columnequilibrated with 10 mM PT. The buffer exchanged material wassubsequently loaded onto a 50×217 mm (˜425 ml) ceramic hydroxyapatitecolumn (Bio-Rad) equilibrated with 10 mM PT. The column was washed with450 ml of the equilibration buffer followed by 900 ml of pH 7.0, 0.25 MNaPO₄ containing 0.1% TX-100. UspA1 was subsequently eluted with alinear NaPO₄ gradient of pH 7.0, 0.25-0.5 M NaPO₄ containing 0.1%TX-100. The fractions containing UspA1 were identified by SDS-PAGE andpooled.

SDS-PAGE and Western blot Analysis. SDS-PAGE was carried out asdescribed by Laemmli (1970) using 4 to 20% (w/v) gradient acrylamidegels (Integrated Separation Systems (ISS), Natick, Mass.). Proteins werevisualized by staining the gels with Coomassie Brilliant Blue R250. Gelswere scanned using a Personal Densitometer SI (Molecular Dynamics Inc.,Sunnyvale, Calif.) and molecular weights were estimated with theFragment Analysis software (version 1.1) using the prestained molecularweight markers from ISS as standards. Transfer of proteins topolyvinylidene difluoride (PVDF) membranes was accomplished with asemi-dry electroblotter and electroblot buffers (ISS). The membraneswere probed with protein specific antisera or MAb's followed by goatanti-mouse alkaline phosphatase conjugate as the secondary antibody(BioSource International, Camarillo, Calif.). Western blots weredeveloped with the BCIP/NBT Phosphatase Substrate System (Kirkegaard andPerry Laboratories, Gaithersburg, Md.).

Protein Estimation. Protein concentrations were estimated by the BCAassay (Pierce, Rockford, Ill.), using bovine serum albumin as thestandard.

Enzymatic and Chemical Cleavages of UspA2 and UspA1.

(i) CNBr Cleavage. Approximately 0.3 mg of the purified protein wasprecipitated with 90% (v/v) ethanol and the pellet resuspended in 100 μlof 88% (v/v) formic acid containing 12 M urea. Following resuspension,100 μl of 88% (v/v) formic acid containing 2 M CNBr (Sigma, St. Louis,Mo.) was added and the mixture incubated overnight at room temperaturein the dark.

(ii) Trypsin and Chymotrypsin Cleavage. Approximately 2 mg of thepurified protein was precipitated with 90% (v/v) ethanol and the pelletresuspended in a total volume of 1 ml of phosphate-buffered saline (PBS)containing 0.1% TX-100. This preparation was added directly to a vialcontaining 25 μg of either trypsin or chymotrypsin (Boehringer Mannheim,Indianapolis, Ind.). The reaction mixture was incubated for 48 h at 37°C.

(iii) Endoproteinase Lys-C Cleavage. Approximately 2 mg of the purifiedprotein was precipitated with 90% (v/v) ethanol and the pelletresuspended in a total volume of 1.0 ml of PBS containing 0.1% TX-100.This preparation was added directly to a vial containing 15 μg ofendoproteinase Lys-C (Boehringer Mannheim). The reaction mixture wasincubated for 48 h at 37° C.

(iv) Separation of Peptides. The above cleavage reaction mixtures werecentrifuged in an Eppendorf centrifuge at 12,000 rpm for 5 min and thesupernatant loaded directly onto a Vydac Protein C4 HPLC column (TheSeparations Group, Hesperia, Calif.). The solvents used were 0.1% (v/v)aqueous trifluoroacetic acid (TFA) [Solvent A] and acetonitrile:H₂0:TFA,80:20:0.1 (v/v/v) [Solvent B] at a flow rate of 1.0 ml/min. Followingthe initial wash with Solvent A, the peptides were eluted with a lineargradient between 0 and 100% of Solvent B and detected by absorbance at220 nm. Suitable fractions were collected, dried in a Speed-Vacconcentrator (Jouan Inc., Winchester, Va.) and resuspended in distilledwater. The fractions were separated by SDS-PAGE in 10 to 18% (w/v,acrylamide) gradient gels (ISS) in a Tris-Tricine buffer system(Schägger and von Jagow, 1987). The fractions containing a singlepeptide band were submitted directly for N-terminal sequence analysis.Fractions displaying multiple peptide bands in SDS-PAGE wereelectrophoretically transferred onto a PVDF membrane as described above.The membrane was stained with Coomassie Brilliant Blue R-250 and theindividual bands excised before submitting them for N-terminal sequenceanalysis (Matsudaira, 1987).

Determination of subunit size. Determination of molecular weight byMatrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF)mass spectrometry (Hillenkamp and Karas, 1990) was done on a Lasermat2000 Mass Analyzer (Finnigan Mat, Hemel Hempstead, UK) with3,5-dimethoxy-4-hydroxy-cinnamic acid as the matrix. Cold ethanolprecipitation was done on samples containing ≦0.1% (v/v) TX-100 toremove the detergent. The final ethanol concentration was 90% (v/v). Theprecipitated protein was resuspended in water.

Determination of aggregate sizes by gel filtration chromatography.Approximately 1 mg of the purified protein was precipitated with 90%(v/v) ethanol and the pellet resuspended in a total volume of 1.0 ml ofPBS containing 0.1% TX-100. Two hundred microliters of the preparationwere applied to a Superose-6 HR 10/30 gel filtration column (10×30 mm,Pharmacia) equilibrated in PBS/0.1% TX-100 at a flow rate of 0.5 ml/min.The column was calibrated using the HMW Calibration Kit (Pharmacia)which contains aldolase with a size of 158,000, catalase with a size of232,000; ferritin with a size of 440,000; thyroglobulin with a size of669,000; and blue dextran with sizes between 2000 and 2,000,000.

Amino Acid Sequence Analysis. N-terminal sequence analysis was carriedout using an Applied Biosystems Model 477A Protein/Peptide Sequencerequipped with an on-line Model 120A PTH Analyzer (Applied Biosystems,Foster City, Calif.). The phenylthiohydantoin (PTH) derivatives wereidentified by reversed-phase HPLC using a Brownlee PTH C-18 column(particle size 5 μm, 2.1 mm i.d.×22 cm 1; Applied Biosystems).

Immunizations. Female BALB/c mice (Taconic Farms, Germantown, N.Y.), age6-8 weeks, were immunized subcutaneously with two doses of UspA1 orUspA2 four weeks apart To prepare the vaccine, purified UspA1 or UspA2was added to aluminum phosphate, and the mixture rotated overnight at4-C. 3-O-deacylated monophosphoryl lipid A (MPL) (Ribi ImmunoChemResearch, Inc.) was added just prior to administration. Each dose ofvaccine contained 5 μg of purified protein, 100 μg of aluminum phosphateand 50 μg of MPL resuspended in a 200 μl volume. Control mice wereinjected with 5 μg of CRM₁₉₇ with the same adjuvants. Serum samples werecollected before the first vaccination and two weeks after the secondimmunization. Mice were housed in a specific-pathogen free facility andprovided water and food ad libitum.

Monoclonal antibodies. The 17C7 MAb was secreted by a hybridoma (ATCCHB11093). MAbs 13-1, 29-31, 45-2, and 6-3 were prepared as previouslydescribed (Chen et al., 1995).

Murine model of M. catarrhalis pulmonary clearance. This model wasperformed as described previously (Chen et al., 1995).

Enzyme linked immunosorbent assay (LISA) procedures. Two different ELISAprocedures were used. One was used to examine the reactivity of sera towhole bacterial cells and the other the reactivity to the purifiedproteins.

For the whole cell ELISA, the bacteria were grown overnight onMueller-Hinton agar and swabbed off the plate into PBS. The turbidity ofthe cells was adjusted to 0.10 at 600 nm and 100 μl added to the wellsof a 96 well Nunc F Immunoplate (Nunc, Roskilde, Denmark). The cellswere dried overnight at 37° C., sealed with a mylar plate sealer andstored at 4° C. until needed. On the day of the assay, the residualprotein binding sites were blocked by adding 5% non-fat dry milk in PBSwith 0.1% Tween 20 (Bovine Lacto Transfer Technique Optimizer [BLOTTO])and incubating 37° C. for one hour. The blocking solution was thenremoved and 100 μl of sera serially diluted in the wells with blotto.The sera were allowed to incubate for 1 h at 37° C. The plate wells weresoaked with 300 ml PBS containing 0.1% Tween 20 for 30 seconds andwashed 3 times for 5 seconds with a Skatron plate washer and thenincubated 1 hr at 37° C. with goat anti-mouse IgG conjugated to alkalinephosphatase (BioSource) diluted 1:1000 in blotto. After washing, theplates were developed at room temperature with 100 μl per well of 1mg/ml p-nitrophenyl phosphate dissolved in diethanolamine buffer.Development was stopped by adding 50 μl of 3N NaOH to each well. Theabsorbance of each well was read at 405 nm and titers calculated bylinear regression. The titer was reported as the inverse of the dilutionextrapolated to an absorption value of 0.10 units.

For the ELISA against the purified proteins, the proteins were dilutedto a concentration of 5 μg/ml in a 50 mM sodium carbonate buffer (pH9.8) containing 0.02% sodium azide (Sigma Chemical Co.). One hundredmicroliters were added to each well of a 96 well E.I.A/R.I.A mediumbinding ELISA plate (Costar Corp., Cambridge, Mass.) and incubated for16 hours at 4° C. The plates were washed and subsequently treated thesame as described for whole cell ELISA procedure.

Complement-dependent bactericidal assay. For this assay, 20 μl of thebacterial suspension containing approximately 1200 cfu bacteria in PBSsupplemented with 0.1 mM CaCl₂, MgCl₂ and 0.1% gelatin (PCMG) were mixedwith 20 μl of serum diluted in PCMG and incubated for 30 min at 4° C.Complement, prepared as previously described (Chen et al., 1996), wasadded to a concentration of 20%, mixed, and incubated 30 min at 35° C.The assay was stopped by diluting with 200 μl of cold, 4° C., PCMG. 50μl of this suspension was spread onto Mueller-Hinton plates. Relativekilling was calculated as the percent reduction in cfu in the samplerelative to that in a sample in which heat inactivated complementreplaced active complement.

Inhibition of bacterial adherence to HEp-2 cells. The effect of specificantibodies on bacterial adherence to HEp-2 cells was examined. A totalof 5×10⁴ HEp-2 cells in 300 μl of RPMI-10 were added to a sterile 8-wellLab-Tek chamber slide Nunc, Inc., Naperville, Ill.) and incubatedovernight in a 5% CO₂ incubator to obtain a monolayer of cells on theslide. The slide was washed with PBS and incubated with 300 μl ofbacterial suspension (A₅₅₀=0.5) or with a bacterial suspension that hadbeen incubated with antisera (1:100) at 37° C. for 1 h. The slides werethen washed with PBS and stained with the Difco quick stain followingthe manufacturers instructions. The slide was viewed and photographedusing a light microscope equipped with a camera (Nikon Microphot-SA,Nikon, Tokyo, Japan).

Protein interaction with fibronectin and vitronectin. The interactionsof purified UspA1 and UspA2 with fibronectin were examined by dot blot.Human plasma fibronectin (Sigma Chemical Co., St. Louis, Mo.) wasapplied to a nitrocellulose membrane, and the membrane blocked withblotto for 1 h at room temperature. The blot was then washed with PBSand incubated with purified UspA1 or UspA2 (2 μg/ml in blotto) overnightat 4° C. After three washes with PBS, the membrane was incubated withthe MAb 17C7 diluted in blotto for 2 h at room temperature and then withgoat anti-mouse immunoglobulin conjugated to alkaline phosphatase(BIO-RAD Lab. Hercules, Calif.) (1:2,000 in PBS with 5% dry milk, 2 h,room temperature). The membrane was finally developed with a substratesolution containing nitroblue tetrazolium and 5-bromo-chloro-3-indolylphosphate in 0.1 M tris-HCl buffer (pH 9.8).

Interaction with vitronectin was examined by a similar procedure. Thepurified UspA1 and UspA2 were spotted onto the nitrocellulose membraneand the membrane blocked with blotto. The membrane was then incubatedsequentially with human plasma vitronectin (GIBCO BRL, Grand, Island,N.Y., 1 μg/ml in blotto), rabbit anti-human vitronectin serum (GIBCOBRL), goat anti-rabbit IgG-alkaline phosphatase conjugate and substrate.

Interaction with HEp-2 cells by the purified protein. Each well of a 96well cell culture plate (Costar Corp., Cambridge, Mass.) was seeded with5×10⁴ HEp-2 cells in 0.2 ml RPMI containing 10% fetal calf serum and theplate incubated overnight in a 37° C. incubator containing 5% CO₂.Purified UspA1 or UspA2 (1 to 1,000 ng) in blotto was added andincubated at 37° C. for 2 h. The plate was washed with PBS, andincubated with the 1:1 mixed mouse antisera to either UspA1 or UspA2(1:1000 dilution in PBS containing 5% dry milk), the plate was washedand incubated with rabbit anti-mouse IgG conjugated to horseradishperoxidase (1:5,000 in PBS containing 5% dry milk) (BrookwoodBiomedical, Birmingham, Ala.) at room temperature for 1 h. Finally, theplate was washed and developed with a substrate solution containing2,2′-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) at 0.3 mg/ml inpH 4.0 citrate buffer containing 0.03% hydrogen peroxide (KPL,Gaithersburg, Md.). Whole bacteria of strain O35E were included as apositive control. The highest concentration of the bacteria tested hadan optical density of A₅₅₀=1.0. The abscissa for the bacterial datashown in FIG. 7 plots the values for three fold dilutions of thebacterial suspension.

Results

Purification of UspA1 and UspA2. The inventors developed a large-scale,high yield process for extracting and purifying UspA2 from a pellet ofM. catarrhalis cells. The method consisted of three critical steps.First the UspA2 protein was extracted from the bacteria with pH 8.0,0.03 M THT. Second, the cell extract was applied to a TMAE column andthe UspA2 protein eluted with NaCl. Finally, the enriched fractions fromthe TMAE chromatography were applied to a ceramic hydroxyapatite columnand the UspA2 eluted with a linear NaPO₄ gradient. A yield of 250 mg ofpurified UspA2 was typically obtained from ˜400 g wet weight of M.catarrhalis O35E strain cells. A single band was seen for the UspA2 inSDS-PAGE gels by Coomassie blue staining. It corresponded to a molecularsize of ˜240,000 and contained greater than 95% of the protein based onscanning densitometry (FIG. 6A). A second band reacting with the 17C7MAb at approximately 125,000 could be detected in the UspA2 preparationby western but not by Coomassie blue staining (FIG. 6C). The cells neednot be lysed to achieve this high yield, which suggested this protein ispresent in large amounts on the surface of the bacterium.

A method for the purification of the UspA1 protein was also developed.This protein co-purified with UspA2 through the initial extraction andTMAE chromatography steps. Following hydroxyapatite chromatography,however, UspA1 remained bound to the column and had to be eluted at thehigher salt concentration of 500 mM NaPO₄. The crude UspA1 preparationobtained in this step was reapplied and eluted from the hydroxyapatitecolumn using a linear sodium phosphate gradient. A total of 80 mg ofpurified UspA1 was isolated from ˜1.6 kg wet wt. of M. catarrhalis O35Estrain cells. UspA1 purified using this method migrated at threedifferent apparent sizes on SDS-PAGE depending on the method of samplepreparation. Unheated samples exhibited a single band at ˜280,000,whereas samples heated at 100° C. for 3 min resulted in an apparentmolecular weight shift to ˜350,000. Prolonged heating at 100° C.resulted in a shift of the 350,000 band to one at 100,000 (FIG. 6B).Following heating of the sample for 7 min at 100° C., the band at100,000 contained greater than 95% of the protein based on scanningdensitometry of the Coomassie stained gel. In contrast, UspA2 migratedat 240,000 regardless of the duration of the heating when examined bySDS-PAGE. The different migration behaviors indicated the preparationscontained two distinctly different proteins

Molecular Weight Determinations. MALDI-TOF mass spectrometric analysisfor determination of molecular weight of UspA2 using3,5-dimethoxy-4-hydroxy-cinnamic acid matrix in presence of 70% (v/v)aqueous acetonitrile and 0.1% TFA resulted in the identification of apredominant species with average-molecular mass of 59,518 Da. Inaddition to the expected [M+H]⁺ and [M+2H]²⁺ molecular ions, the [2M+H]⁺and [3M+H]⁺ ions were also observed. The latter two ions were consistentwith the dimer and the trimer species. Using similar conditions, theinventors were unable to determine the mass of UspA1.

To determine the molecular sizes of the purified proteins in solution,UspA1 and UspA2 were independently run on a Superose-6 HR 10/30 gelfiltration column (optimal separation range: 5,000-5,000,000) calibratedwith molecular weight standards. Purified UspA1 exhibited a nativemolecular size of 1,150,000 and UspA2 a molecular size of 830,000. Thesesizes, however, may be affected by the presence of TX-100.

N-terminal Sequence Analysis of Internal UspA1 and UspA2 Peptides. Allattempts to determine the N-terminal sequences of both UspA and UspA1proved unsuccessful. No sequence could be determined. This suggested twothings. First, the N-terminus of both proteins were blocked, and,second, neither protein preparation contained contaminating proteinsthat were not N-terminally blocked.

Thus, to confirm that the primary sequence of purified UspA1 and UspA2corresponded to that deduced from their respective gene sequences,internal peptide fragments were generated and subjected to N-terminalsequence analysis. Tables X and XI show the N-terminal sequencesobtained for fragments generated from the digestion of the UspA2 andUspA1 proteins, respectively. The sequences matching the primary aminoacid sequence deduced from the respective gene sequences are indicatedfor each fragment. The UspA2 fragments #3 and #4 exhibited sequencesimilarity with residues 505-515 and 605-614 respectively of the aminoacid sequence deduced from the UspA1 gene. In Table XII, UspA1 fragment#3 exhibited sequence similarity with residues #278-294 of the UspA2primary sequence. These sequences corresponded with the domains withinUspA1 and UspA2 that share 93% sequence identity. The remainder of thesequences, however, were unique to the respective proteins

TABLE X N-terminal sequences of internal UspA2 peptide cleavagefragments UspA2 Fragment Sequence^(a) Match^(b) Cleavage 1) LLAEQQLNG 92-100 Trypsin SEQ ID NO:74 2) ALESNVEEGL 216-225 Lys-C SEQ ID NO:74245-254 274-283 3) ALESNVEEGLLDLS 274-288 Trypsin SEQ ID NO:75 *505-515 4) AKASAANTDR 378-387 Chymotrypsin SEQ ID NO:76 *605-614  5)AATAADAITKNGN 439-450 Chymotrypsin SEQ ID NO:77 6) SITDLGTKVDGFDGR458-472 Lys-C SEQ ID NO:78 7) VDALXTKVNALDXKVN 473-488 Trypsin SEQ IDNO:79 8) AAOAALSGLFVPYSVGKFNATAAL 506-535 CNBr GGYGSK SEQ ID NO:80^(a)Underlined residues denote mismatch with the nucleotide derivedamino acid sequence. Ambiguous residues whose identity could not beverified are denoted by the letter X. ^(b)Asterisk (*) indicates matchwith UspA1. Without asterisk indicates matches with nucleotide derivedamino acid sequence of UspA2.

TABLE XI N-terminal sequences of internal UspA1 peptide cleavagefragments UspA1 Fragment Sequence^(a) Match^(b) Cleavage 1)LENNVEEPXLNLS 456-468 Lys-C 2) DQKADI 473-478 Trypsin 3)NNVEEGLLDLSGRLIDQK 504-521 Lys-C *278-294  4) VAEGFEIF 690-697 Trypsin5) AGIATNKQELILQNDRLNRI 701-720 Lys-C ^(a)As per Table X. X denotes anunidentified amino acid residue. ^(b)Asterisk (*) indicates match withUspA2. Without asterisk indicates matches with nucleotide derived aminoacid sequence of UspA1.

Reactivity of MAbs with UspA1 and UspA2. The western blot analysis ofpurified UspA1 and UspA2 revealed that both proteins reacted stronglywith the MAb 17C7 described by Helminen et al. (1994) (FIG. 7). Thereactivity of the proteins with other MAbs was also investigated. Thedata in Table XII show that, whether assayed by ELISA or western, theMAbs 13-1, 29-31 and 45-2 only reacted with UspA2, the MAbs 7D7, 29C6,11A6 and 12D5 only reacted with UspA1, while 17C7 and 6-3 reacted withboth UspA1 and UspA2. All the MAbs shown in Table XIII bind to wholebacteria when examined by ELISA. These results indicated that UspA2 wasexposed on the surface of the bacterium.

TABLE XII Summary of reactivity of monoclonal antibodies with purifiedUspA1, UspA2 and whole bacteria of strain O35E Reactivity Whole PurifiedmAb Isotype bacterium^(a) UspA1^(b) Purified UspA2^(b) 13-1 IgG1κ + − +29-31 IgG1λ + − + 45-2 IgG2a + − + 17C7 IgG2a + + + 6-3 IgM + + + 7D7IgG2b + + − 29C6 IgG1 + + − 11A6 IgA + + − 12D5 IgG1 + + −^(a)Determined by whole cell ELISA. ^(b)Determined by ELISA and westernblot.

TABLE XIII Cross-reactivity of antibodies to UspA1 and UspA2 proteinsGeometric mean ELISA titer^(b) to Antiserum to UspA1 UspA2 UspA1^(a)740,642^(c) 10,748^(c) UspA2^(a)  19,120^(d) 37,615^(d) ^(a)Thepreparation of the sera are described in the text. ^(b)ELISA titers arefor total IgG and IgM antibodies for sera pooled from ten mice. ^(c)Thedifference in titer of the anti-UspA1 with the two purified proteins wasstatistically different by the Wilcoxon signed rank test (p = 0.0002).^(d)The difference in titer of the anti-UspA2 with the two purifiedproteins was statistically different by the Wilcoxon signed rank test (p= 0.01).

Immunogenicity and antibody cross-reactivity. Antisera to the purifiedUspA1 and UspA2 proteins were generated in mice. The titers of antigenspecific antibodies (IgG and IgM) as well as the cross-reactiveantibodies in these sera were determined by an ELISA assay using each ofthe purified proteins (Table XIII). Both proteins elicited antibodytiters that were greater against themselves than against theheterologous protein. Thus, the reactivities of both the MAbs (TableXII) as well as the polyclonal antibodies indicate that the proteinspossessed both shared and non-shared B-cell epitopes.

Antibody reactivity to whole bacterial cells and bactericidal activity.Antisera to the UspA1 and UspA2 were assayed by whole cell ELISA againstthe homologous O35E strain and several heterologous isolates (TableXIV). The antibodies to UspA1 and to UspA2 reacted strongest with theO35E stain. The reactivity of the sera toward the heterologous isolatesindicated they bound antibodies elicited by both UspA1 and UspA2.

TABLE XIV ELISA and complement mediated bactericidal titers toward wholebacterial cells of multiple isolates of M. catarrhalis elicited bypurified UspA1 and purified UspA2 Whole cell ELISA^(a) Bactericidaltiter^(b) Isolate anti-UspA1^(a) anti-UspA2^(a) anti-UspA1 anti-UspA2O35E 195,261 133,492 400 800 430-345 12,693 18,217 400 400 1230-3597,873 13,772 400 400 TTA24 14,341 7,770 800 800 ^(a)Titer determined forpool of sera from ten mice. The titer of the sera drawn before the firstimmunization was less than 50 for all isolates. ^(b)Bactericidal titerswere determined as the inverse of the highest serum dilution killinggreater than 50% of the bacteria. The titers for the sera from miceimmunized contemporaneously with CRM₁₉₇ were less than 100.

The bactericidal activities of the antisera to UspA1 and UspA2 weredetermined against O35E and other isolates as well (Table XIV). Bothsera had bactericidal titers ranging from 400-800 against O35E and thedisease isolates. Anti-CRM₁₉₇ serum, the negative control, as well assera drawn before immunization, had a titers of <100 against all thestrains. These results were consistent with the previous observationthat the epitopes shared by the two proteins are highly conserved amongisolates and the antibodies toward those isolates are bactericidal.

Pulmonary challenge. Immunized mice were given a pulmonary challengewith the homologous O35E strain or the heterologous TTA24 strain.Relative to the control mice immunized with CRM₁₉₇, enhanced clearanceof both strains was observed regardless of whether the mice wereimmunized with UspA1 or UspA2 (Table XV). No statistical difference(p>0.05) was seen between the groups of mice immunized with UspA1 andwith UspA2.

TABLE XV Pulmonary clearance of M. catarrhalis by mice immunized withpurified UspA1 and UspA2 Study Immunogen Challenge strain %clearance^(a) p^(a) 1 UspA1 O35E 49.0 0.013 UspA2 31.8 0.05 CRM₁₉₇ 0 — 2UspA1 TTA24 54.6 0.02 UspA2 66.6 0.0003 CRM₁₉₇ 0 — ^(a)Challenge methoddescribed in text. Numbers are the percentage of bacteria cleared fromthe immunized mice compared to control mice which were immunized withCRM₁₉₇.

Interaction of purified proteins with HEp-2 cells. The purified UspA1and UspA2 were tested for their ability to interact with HEp-2 cellmonolayer in a 96-well plate using an ELISA. Protein binding to theHEp-2 cells was detected with a 1:1 mix of the mouse antisera to UspA1and UspA2. Purified UspA1 bound to HEp-2 cells at concentrations above10 ng. A weak binding by the UspA2 was detected at concentrations above100 ng (FIG. 7). The attachment of O35E bacteria to HEp-2 cells was usedas a positive control. This result, plus the data showing that theanti-UspA1 antibodies inhibited attachment of the bacteria to HEp2cells, suggests UspA1 plays an important role in bacterial-attachmentwhich also suggested that UspA1 was exposed on the bacterial surface.

Interaction of purified proteins with fibronectin and vitronectin. Thepurified proteins were assayed for their ability to interact withfibronectin and vitronectin by dot blot assays. Human plasma fibronectinimmobilized on a nitrocellulose membrane bound purified UspA1 but notUspA2 (FIG. 8), while UspA2 immobilized on the nitrocellulose membranewas capable of binding vitronectin (FIG. 8). Vitronectin binding by theUspA1 was also detected, but the reactivity was weaker. Collagen (typeIV), porcine mucin (type III), fetuin and heparin were also tested forinteraction with purified UspA1 and purified UspA2, but these did notexhibit detectable binding.

Discussion

Previous UspA purification attempts yielded preparations containingmultiple high molecular weight protein bands by SDS-PAGE and westernblot. Because each of the bands reacted with the “UspA specific” MAb17C7, it was thought they represented multiple forms of the UspA protein(Chen et al., 1996). However, the inventors have discovered that thereare two distinct-proteins, UspA1 and UspA2, that share an epitoperecognized by the 17C7 MAb. These two proteins are encoded by differentgenes. This study shows that UspA1 and UspA2 can be separated from oneanother. The isolated proteins had different SDS-PAGE mobilitycharacteristics, different reactivity with a set of monoclonalantibodies, and different internal peptide sequences. The results,however, were consistent with the proteins sharing a portion of theirpeptide sequences, including the MAb 17C7 epitope. The separation of theproteins from one another has allowed the inventors to furtherdemonstrate how the proteins were different as well as examine theirbiochemical, functional, and immunological characteristics.

In solution, the purified proteins appear to be homopolymers of theirrespective subunits held together by strong non-covalent forces. This isindicated by the fact that UspA2 lacks any cysteines and treatment ofboth proteins with reducing agents did not alter their mobilities inSDS-PAGE. Both gene sequences possess leucine zipper motifs that mightmediate coil-coil interactions (O'Shea et al., 1991). Even so, it wassurprising that the non-covalent bonds of both proteins were not onlystrong enough to resist dissociation by the conditions normally used toprepare samples for SDS-PAGE, but also high concentrations of chaotropicagents such as urea (Klingman and Murphy, 1994) and guanidine HCl. Ofthe two proteins, UspA2 appeared to be less tightly aggregated, this wasindicated by the fact that its subunit size of 59,500 Da could bedetermined by mass spectrometry. UspA1, however, was recalcitrant todissociation by all the methods tried, and this may be the reason itssize could not be determined by mass spectrometry. In SDS-PAGE, thedominant UspA2 migrated with an apparent size of 240,000 while a farsmaller portion migrated at about 125,000 and could only be detected bywestern analysis. The mobility of UspA1, however, varied depending onhow long the sample was heated. The smallest form was about 100,000.This was consistent with the size of the gene product missing from theuspA1 mutant but not with the size predicted from the gene sequence of88,000 Da. In solution, both proteins formed larger aggregates thanthose seen by SDS-PAGE. Their sizes, as measured by gel filtrationchromatography, were 1,150,000 and 830,000 for UspA1 and UspA2respectively. If the proteins behave this way in vivo, UspA1 and UspA2likely occur as large molecular complexes on the bacterial surface ofthe bacterium.

The results of the N-terminal amino acid sequence analyses of the UspA2and UspA1 derived peptides (Tables X and XI) were in agreement with theprotein sequences derived from the respective gene sequences. Thisconfirmed that the purified UspA1 and UspA2 proteins were the productsof the respective uspA1 and uspA2 genes. Further, the experimental andtheoretical amino acid compositions of UspA1 and UspA2 were consistent,given the size of the proteins and the accuracy of the amino aciddetermination. There was, however, a discrepancy between the sizedetermined by mass spectrometry of 59,518 and the size indicated fromthe gene sequence for UspA2 of 62,483. This discrepancy suggested thatthis protein either undergoes post-translational processing orproteolytic degradation.

The data also suggest that both proteins are exposed on the bacterialsurface. That at least one of the proteins is exposed is evident fromthe finding that the MAb 17C7 and polyclonal sera react with wholecells. The reactivities of the UspA2 specific monoclonal antibodies13-1, 29-31 and 45-2 with the bacterial cells in the whole cell ELISAprovided evidence that the UspA2 is a surface protein (Table XII). Thereactivities of the UspA1 specific MAbs 7D7, 29C6, 11A6 and 12D5 withthe bacterial cells in the whole cell ELISA provided evidence that theUspA1 is a surface protein (Table XII). Further evidence for surfaceexposure of UspA1 was indicated by the inhibitory effect of theantiserum on bacterial attachment to HEp-2 cells. The sera to the UspA2lacked this activity. Thus, both UspA1 and UspA2 appeared to be surfaceexposed on the bacterium.

Surface exposure of the proteins is probably important for the twoproteins' functions. One function for UspA1 appears to be meditation ofadherence to host tissues. The evidence for this was that UspA1antibodies inhibited bacterial binding to HEp-2 cells and the purifiedprotein itself bound to the cells. The relevance of binding to HEp-2cells is that they are epithelial cells derived from the larynx, acommon site of M. catarrhalis colonization (Schalen et al., 1992). Thisconfirms the inventors findings that mutants that do not express UspA1fail to bind epithelial cells. The inventors' also showed that UspA1binds fibronectin. Fibronectin has been reported to be a host receptorfor other pathogens (Ljungh and Wadström, 1995; Westerlund and Korhonen,1993). Examination of the gene sequence, however, failed to reveal anysimilarity with the fibronectin binding motifs reported for Grampositive organisms (Westerlund and Korhonen, 1993). Thus, it is fairlyclear that UspA1 plays a role in host adherence, possibly via cellassociated fibronectin.

The function of UspA2 is less certain. Antibodies toward it did notblock adherence to the HEp-2 or Chang cell lines, nor did the purifiedprotein bind to those cells. Yet, UspA2 bound vitronectin strongly.Pathogen binding of vitronectin has been linked to host cell adherence(Gomez-Duarte et al., 1997; Limper et al., 1993); however, van Dijk andhis co-workers have reported that vitronectin binding by M. catarrhalismay be used by the bacteria to subvert host defenses (Verdiun et al.,1994). The soluble form of vitronectin, known as complement factor S,regulates formation of the membrane attack complex (Su, 1996). Theysuggest that the binding of vitronectin to the M. catarrhalis surfaceinhibits the formation of the membrane attack complex, rendering thebacteria resistant to the complement dependent killing activity of thesera. They have also described two types of human isolates: one thatbinds vitronectin and is resistant to the lytic activity of the serumand the other that does not bind vitronectin and is serum sensitive (Holet al., 1993). It must be noted, however, that vitronectin, like all theextracellular matrix proteins, has many forms and serves multiplefunctions in the host (Preissner, 1991; Seiffert, 1997). Thus, theinteraction of both UspA1 and UspA2 with the extracellular matrixproteins fibronectin and vitronectin may serve the bacterium in waysbeyond subverting host defenses or as receptors for bacterial adhesion.

Even though the two proteins share epitopes and sequences they havedifferent biochemical activities and likely serve different biologicalfunctions. If an immune response to the respective protein interfereswith its function, it ought to be considered as a vaccine candidate. Theresults of the immunological studies in mice indicated that bothproteins would be good vaccine candidates. Mice immunized with eitherUspA1 or UspA2 developed high antibody titers toward the homologous andheterologous bacterial isolates. Further, the sera from these mice hadcomplement dependent bactericidal activity toward all the isolatestested. In addition, immunized mice exhibited enhanced pulmonaryclearance of the homologous isolate and heterologous isolates. It isimportant to note that antibodies elicited by the proteins werepartially cross-reactive. This was expected since both react with the17C7 MAb and share amino acid sequence.

Example V The Level and Bactericidal Capacity of Child and Adult HumanAntibodies Directed Against the Proteins UspA1 and UspA2

To determine if humans have naturally acquired antibodies to the UspA1and UspA2 of the M. catarrhalis and the biological activity of theseantibodies if present, sera from healthy humans of various ages wasexamined using both ELISA and a bactericidal assay. It was found thathealthy people have naturally acquired antibodies to both UspA1 andUspA2 in their sera, and the level of these antibodies and theirbactericidal capacity were age-dependent. These results also indicatethat naturally acquired antibodies to UspA1 and UspA2 are biologicallyfunctional, and thus support their use as vaccine candidates to preventM. catarrhalis disease.

Material and Methods

Bacteria. The M. catarrhalis strains O35E and TTA24 were as described inExample 1. An ATCC strain (ATCC 25238) and three other clinical isolatesfrom the inventors' collection were also used.

Human sera. Fifty-eight serum samples were collected from a group of tenchildren at 2, 4, 6, 7, 15 and 18 months of age who had received routinechildhood immunizations. Individual sera from twenty-six adults andfifteen additional children 18-36 months of age were also assayed. Allsera were obtained from clinically healthy individuals. Information onM. catarrhalis colonization and infection of these subjects was notcollected. The sera were stored at −70° C.

Purification of UspA1 and UspA2. Purified UspA1 and UspA2 were made fromthe O35E strain of M. catarrhalis as described in Example IV herein.Each protein preparation contained greater than 95% of the specificprotein based on densitometric scanning of Coomassie brilliant bluestained SDS-PAGE. Based on western blot analysis using monoclonalantibodies, each purified protein contained no detectable contaminationof the other.

Purification of UspA1 and UspA2 specific antibodies from human plasma.Human plasmas from two healthy adults were obtained from the AmericanRed Cross (Rochester, N.Y.) and pooled. The antibodies were precipitatedby adding ammonium sulfate to 50% saturation. The precipitate wascollected by centrifugation and dialyzed against PBS. A nitrocellulosemembrane (2×3 inches) was incubated with UspA1 or UspA2 at 0.5 mg/ml inPBS containing 0.1% (vol/vol) Triton X-100 for 1 h at room temperature,washed twice with PBS and residual binding sites on the membrane blockedwith 5% (wt/vol) dry milk in PBS for 2 h at room temperature. Themembrane was then sequentially washed twice with PBS, 100 mM glycine (pH2.5) and finally with PBS before incubation with the dialyzed antibodypreparation. After incubating for 4 h at 4° C., the membrane was washedagain with PBS, and then 10 mM Tris buffer (pH 8.0) containing 1 Msodium chloride to remove non-specific proteins. The bound antibodieswere eluted by incubation in 5 ml of 100 mM glycine (pH 2.5) for 2 minwith shaking. One ml of Tris-HCl (1M, pH 8.0) was immediately added tothe eluate to neutralize the pH. The eluted antibodies were dialyzedagainst PBS and stored at −20° C.

Enzyme-linked immunosorbent assay (ELISA). Antibody titers to the O35Eand other M. catarrhalis strains were determined by a whole-cell ELISAas previously described using biotin-labeled rabbit anti-human IgG orIgA antibodies (Brookwood Biomedical, Birmingham, Ala.) (Chen et al.,1996). Antibody titers to UspA1 and UspA2 were determined by a similarmethod except that the plates were coated with 0.1 μg of purifiedprotein in 100 μg of PBS per well overnight at room temperature. The IgGsubclass antibodies to UspA1 or UspA2 were determined using sheepanti-human IgG subclass antibodies conjugated to alkaline phosphatase(The Binding Site Ltd., San Diego, Calif.). The antibody end point titerwas defined as the highest serum dilution giving an A₄₁₅ greater thanthree times that of the control. The control wells received alltreatments except human sera and usually had absorbance values rangingfrom 0.03 to 0.06.

The specificity of biotin-labeled rabbit anti-human IgG and IgAantibodies was determined against purified human IgG, IgM and IgA(Pierce, Rockford, Ill.) by ELISA. No cross-reactivity was found. Theassay sensitivity determined by testing against purified humanantibodies of appropriate isotype in an ELISA was 15 and 60 ng/ml in theIgG and IgA assays, respectively. Likewise, the specificity of the humanIgG subclass antibody assays was confirmed in ELISA against purifiedhuman myeloma IgG subclass proteins (ICN Biomedicals, Inc., Irvine,Calif.), and the assay sensitivity was 15 ng/ml in the IgG1, IgG3 andIgG4 assays, and 120 ng/ml in the IgG2 assay. Two control sera wereincluded to control for assay to assay variation.

Complement dependent bactericidal assay. The bactericidal activity ofthe human sera was determined as described previously (Chen et al.,1996). In some studies, the sera were absorbed with purified UspA1 orUspA2 prior to the assay. The absorption of specific antibodies fromthese sera was accomplished by adding the purified proteins to 20 or 50μg/ml final concentration. The final serum dilution was 1:10. Themixtures were incubated for 2 h at 4° C. and the precipitate removed bymicro-centrifugation. The purified human antibodies specific for UspA1and UspA2 were assayed against five M. catarrhalis strains in a similarmanner.

Statistics. Statistical analysis was performed on logarithmictransformed titers using JMP software (SAS institute, Cary, N.C.). Toallow transformation, a value of one half the lowest serum dilution wasassigned to sera which contained no detectable titers. Comparison of IgGlevels among the age groups was done by analysis of variance, and therelationship of antibody titer and the bactericidal titer was determinedby logistic regression. A p value less than 0.05 was consideredsignificant.

Results

Comparison of serum IgG and IgA titers to UspA1 and UspA2 in childrenand adults. The IgG and IgA antibody titers in the sera from tenchildren collected longitudinally between 2-18 months of age, as well asthe random samples from fifteen 18-36 month old children and twenty-sixadults were determined against the whole bacterial cells of the O35Estrain, the purified UspA1 and the purified UspA2 by ELISA. IgG titersto all three antigens were detected in almost all the sera (FIG. 9). TheIgG titers to UspA1 and UspA2 exhibited strong age-dependent variationwhen compared to IgG titers to the O35E bacterium (FIG. 9). The adultsera had significantly higher IgG titers to the purified proteins thansera from children of various age groups (p<0.01). Sera from children at6-7 months of age had the lowest IgG titers to UspA proteins and themean titer at this age was significantly lower than that at 2 months ofage (p<0.05).

The level of IgA antibodies to UspA1, UspA2 and O35E bacterial cellswere age dependent (FIG. 9). A serum IgA titer against the UspA1 andUspA2 was detected in all twenty-six adults and children of 18-36 monthsof age. For children less than 18 months of age, the proportionexhibiting antigen specific IgA titers increased with age. The mean IgAtiters to UspA1, UspA2 or O35E bacterium in these sera were low for thefirst 7 months of age but gradually increased thereafter (FIG. 9).

Age-dependent subclass distribution of IgG antibodies to UspA1 andUspA2. The IgG subclass titers to the UspA1 and UspA2 antigens weredetermined on sera from ten adult sera and thirty-five children's sera.The subclass distribution was found to be age-dependent. The mostprominent antibodies to the UspA1 and UspA2 antigens were of the IgG1and IgG3 subclasses, which were detected in almost all sera. The IgG2and IgG4 titers were either undetectable or extremely low. Therefore,only data on IgG1 and IgG3 subclasses are reported (FIG. 10). The IgG3titers against UspA1 or UspA2 in the adult sera were significantlyhigher than the IgG1 titers (p<0.05). The same subclass profile was seenin the sera from the 2 month old children, although the differencebetween IgG1 and IgG3 titers did not reach statistical significance,probably because of the smaller sample size. Sera from children between4 and 36 months of age all had a similar subclass profile which wasdifferent from that of the adults and 2 month old children. The IgG1titers in children's sera were either higher than or equivalent to theIgG3 titers. The mean IgG1 titer to either UspA1 or UspA2 wassignificantly higher than IgG3 titer to the same antigens in thesechildren's sera (p<0.05).

Bactericidal activity. The bactericidal titers of seventeen serarepresenting different age groups were determined (Table XVI). All theadult sera and three out of five sera from the two month old childrenwhich had high IgG titers to the UspA proteins had strong bactericidalactivity. Sera from 6 month old children had the least bactericidalactivity. All five sera from this age group had a marginal bactericidaltiter of 50, the lowest dilution assayed. The bactericidal activity ofthe sera from 18 to 36 month old children was highly variable withtiters ranging from less than 50 to 500. There was a significant linearrelationship between the bactericidal titers and the IgG antibody titersagainst both UspA1 and UspA2 by logistic regression analysis (p<0.01)(FIG. 11).

TABLE XVI The level of IgG antibodies to UspA1 and UspA2 from normalhuman serum and the serum bactericidal activity ELISA IgG titer^(b)Subject^(a) Age UspA1 UspA2 BC titer^(c) 1 2 month 17,127 6,268 500 6month 4,273 1,363 50 15 month 798 250 <50 2 2 month 12,078 12,244 500 6month 1,357 878 50 18 month 14,041 14,488 200 3 2 month 30,283 20,362500 6 month 1,077 1,947 50 18 month 2,478 1,475 <50 4 2 month 2,086 869<50 6 month 530 802 50 18 month 9,767 8,591 200 5 2 month 3,233 2,655<50 6 month 2,246 360 50 18 month 26,693 43,703 500 6 1.5-3 year 4,0362,686 50 7 1.5-3 year 2,037 1,251 50 8 1.5-3 year 341 251 <50 9 1.5-3year 2,538 1,200 500 10 1.5-3 year 1078 1,370 500 11 1.5-3 year 1,265953 50 12 adult 161,750 87,180 450 13 adult 873,680 248,290 >1350 14adult 154,650 146,900 450 15 adult 10,330 7,860 50 16 adult 35,78031,230 150 17 adult 19,130 132,200 450 ^(a)Three consecutive samplesfrom subjects 1 through 5 were collected at the stated ages. ^(b)ELISAend point titers to purified UspA1 or UspA2 from the O35E strain weredetermined as the highest serum dilution giving an A₄₁₅ greater thanthree times the background. ^(c)BC titers: bactericidal titer assayedagainst the O35E strain. Sera were assayed at 1:50, 100, 200, and 500.Bactericidal titer was determined as the highest serum dilutionresulting in killing of 50% or more of the bacteria relative to thecontrol. Control bacteria were incubated with test serum and heatinactivated complement serum.

Bactericidal activity of sera absorbed with purified UspA1 or UspA2.Because normal human sera contain antibodies to numerous antigens of M.catarrhalis as indicated by western blot, an absorption method was usedto determine the contribution of UspA1 and UspA2 specific antibodiestowards the bactericidal activity. Six adult sera were absorbed withpurified UspA1 or UspA2, and the change in ELISA reactivity to UspAproteins determined. A reduction in ELISA reactivity was seen for allthe sera after absorption (Table XVII). Further, absorption with oneprotein resulted in a reduction of IgG titers to the other protein.Reduction of UspA2 reactivity was of the same degree regardless ofwhether the absorbent was UspA1 or UspA2. In contrast, there was lessreduction in UspA1 reactivity after absorption with UspA2 than withUspA1 (Table XVII). This indicated that antibodies to UspA1 and UspA2were partially cross-reactive.

TABLE XVII ELISA titer of adult sera before and after absorption^(a) Ab-sorbent #1 #2 #3 #4 #5 #6 IgG titers to UspA1 in sample^(b) saline161,750 873,680 154,650 10,330 35,780 19,130 UspA1 2,450 2,210 3,1601,650 <500 3,010 UspA2 42,620 90,150 33,570 6,420 3,490 4,130 IgG titersto UspA2^(b) saline 87,180 248,290 146,900 7,860 31,230 13,200 UspA12,800 2,120 2,700 2,220 <500 <500 UspA2 <500 1,820 3,010 2,960 <500 <500^(a)Absorption: An aliquot of adult serum was diluted and added withpurified UspA1 or UspA2 from O35E strain to a final 50 μg/ml proteinconcentration and final 1:10 serum dilution. The mixtures were incubatedat 4° C. for 2 h, and precipitates removed by microcentrifugation.^(b)IgG titers against the UspA1 and UspA2 proteins were end pointtiters determined with a starting serum dilution of 1:500.

The bactericidal titers of the absorbed sera were determined andcompared with those seen before absorption (Table XVIII). Absorptionwith either UspA1 or UspA2 resulted in complete loss of bactericidalactivity (<50) for all six sera when assayed against the O35E strain,the strain from which the purified proteins were made (Table XVIII). Thebactericidal activity of the absorbed sera was also reduced by at leastthree fold when assayed against the a heterologous strain 1230-359.Absorption using UspA1 resulted in greater reduction of the bactericidaltiter against the heterologous strain in 3 out of 6 samples compared toabsorptions using UspA2 (Table XVII). This result was consistent withthe difference in the reductions of ELISA titers to the UspA1 afterabsorption with the two proteins. Absorption using the combined proteinsUspA1 and UspA2 did not result in further reduction of the bactericidalactivity compared to UspA1 alone. All six human sera containedantibodies to a 74 kDa OMP from M. catarrhalis as determined by westernblot analysis, and absorption using the purified 74 kDa protein did notaffect the bactericidal activity of either the O35E strain or the1230-357 strain. This indicated that antibodies to the UspA proteinswere the major source of the bactericidal activity against M.catarrhalis in adult sera.

TABLE XVIII Bactericidal titer of the adult human sera before and afterabsorption^(a) Adsorbent #1 #2 #3 #4 #5 #6 Bactericidal titer to O35Estrain in sample^(b) saline 450 >1350 450 50 150 450 UspA1 <50 <50 <50<50 <50 <50 UspA2 <50 150 <50 <50 <50 <50 Bactericidal titer to 1230-359strain^(b) saline 450 4050 >1350 150 150 450 UspA1 50 150 <50 <50 50 150UspA2 150 1350 450 <50 50 50 ^(a)Sera were the same as those describedin Table XVII. ^(b)Bactericidal titer: The bactericidal activity wasmeasured against the O35E or 1230-359 strains with 3-fold diluted serastarting at 1:50. The highest serum dilution resulting in 50% or greaterkilling was determined as the bactericidal titer. The purified UspA1 andUspA2 proteins used for absorption were made from the O35E strain.

Because only small volumes of the children sera were available,absorption of these sera was done using a mixture of UspA1 and UspA2proteins. Absorption resulted in the complete loss or a significantreduction of bactericidal activity in four out of seven sera (TableXIX). The four sera including three from two month old children all hadan initial bactericidal titer of 200 or greater prior to absorption. Theother three sera, which did not show a change in bactericidal titer uponabsorption, all had a marginal titer of 50 before absorption. Thereduction in ELISA reactivity to the UspA proteins after absorptionconfirmed that the antibody concentration had been reduced. Thissuggested that antibodies specific for the UspA1 and UspA2 proteins inchildren's sera were also a major source of the bactericidal activitytowards M. catarrhalis.

TABLE XIX Bactericidal activity of children's sera before and afterabsorption with pooled purified UspA1 and UspA2^(a) Age Unabsorbed serumAbsorbed serum Sample (months) A₄₁₅ ^(b) BC titer^(c) A₄₁₅ ^(b) BCtiter^(c) 1 2 0.84 200 0.29 <50 2 2 0.93 200 0.19 <50 3 2 0.98 500 0.3850 4 18 0.88 200 0.43 50 5 15 0.66 50 0.25 50 6 18 0.62 50 0.32 50 7 150.68 50 0.35 50 ^(a)Absorption: Each serum was absorbed with a mixtureof UspA1 and UspA2 proteins from O35E strain at final proteinconcentrations of 200, 50 or 20 μg/ml. The same result was seen for allthree absorptions of each sample. Only the data from the assay using 20μg/ml of protein are shown. ^(b)A₄₁₅: The absorbance at 415 nm in ELISAusing the mixture of UspA1 and UspA2 as detection antigen. Sera weretested at a 1:300 dilution. ^(c)BC titer: Highest serum dilutionresulting in 50% or greater killing of the O35E strain in the assay.Sera were assayed at dilutions 1:50, 200, and 500.

Affinity purified antibodies to UspA1 and UspA2: To confirm theircross-reactivity and bactericidal activity, antibodies to UspA1 or UspA2from adult plasma were isolated by an affinity purification procedure.The purified antibodies reacted specifically with the UspA1 and theUspA2 proteins but not with non-UspA proteins in the O35E lysates in awestern blot assay. The purified antibodies to one protein also reactedto the other with almost equivalent titer in ELISA (Table XX). Bothantibody preparations exhibited reactivity with five M. catarrhalisstrains in the whole-cell ELISA and bactericidal assay (Table XXI). Thebactericidal titers against all five M. catarrhalis strains rangedbetween 400 and 800, which was equivalent to 0.25-0.50 μg/ml of theprotein in the purified antibody preparations (Table XXI).

TABLE XX Cross-reactivity of affinity purified human antibodies to UspA1and UspA2 in ELISA IgG titers against^(b) Antibodies purified to^(a)UspA1 UspA2 UspA1 50,468 20,088 UspA2 53,106 52,834 ^(a)The antibodieswere purified from plasma pooled from two healthy adults by immuneelution using purified UspA1 or UspA2 from the O35E strain immobilizedon nitrocellulose membrane. ^(b)ELISA end point titers are the highestantibody dilutions giving an A₄₁₅ greater than three times thebackground.

TABLE XXI Whole cell ELISA titer and bactericidal titer of affinitypurified human antibodies to UspA1 and UspA2^(a) Assay Whole cell ELISAtiter^(b) BC titer^(c) strain Ab to UspA1 Ab to UspA2 Ab to UspA1 Ab toUspA2 O35E 12,553 9,939 400 800 ATCC25238 30,843 29,512 400 400 TTA2451,511 57,045 800 800 216:96 31,140 23,109 400 400 1230-359 8,495 16,458800 800 ^(a)The purified antibody preparations were the same asdescribed in Table XX. The specific reactivities of the purifiedantibodies to UspA proteins, but not other outer membrane proteins, wereconfirmed by western blots. ^(b)ELISA end point titers are the highestantibody dilutions giving an A₄₁₅ greater than three times thebackground when assayed against whole bacterial cells. ^(c)BC titer:Highest antibody dilution resulting in 50% or greater killing of thebacterial inoculum in the assay. Antibodies (120 μg/ml) were assayed atdilutions 1:100, 200, 400, and 800.Discussion

Previous studies examining human antibodies to M. catarrhalis wholecells or outer membrane proteins usually focused on a single age group.Further, the biological function of the antibodies was left largelyundetermined (Chapman et al., 1985), and the antigens eliciting thefunctional antibodies were not identified. Thus, these previous studiesdid not provide information as to the role of naturally acquiredantibodies in protection against M. catarrhalis diseases, nor did theyprovide clear information as to what antigens are suitable for vaccinedevelopment. The data from this study indicate that the IgG antibodiesto UspA1 and UspA2 are present in normal human sera and their levels areage-dependent. These antibodies are an important source of serumbactericidal activity in both children and adults.

These data indicated that most children had serum IgG antibodies to bothUspA1 and UspA2 at two months of age although the level varied fromindividual to individual, and the IgG subclass profile in these infantsera was similar to that in adult sera. The infant sera had bactericidalactivity. The absorption studies suggested that the bulk of thebactericidal antibodies in these sera were directed against the UspA1and the UspA2 proteins. These results suggest that the IgG antibodiesdetected in the two month old children are of maternal origin. This isconsistent with the report that umbilical cord serum contains hightiters of antibodies to an extract of M. catarrhalis whole cells(Ejlertsen et al., 1994b).

Due to the lack of clinical information on the study subjects and smallnumber of subjects examined in this study, it could not be determinedwhether maternal antibodies against UspA, although bactericidal invitro, were protective in young children. However, at two months of agethe children had significantly higher serum IgG titers against the UspAproteins and only a few of these children had a low level of IgAantibodies to M. catarrhalis as compared to children at 15-18 months ofage. If serum IgA reflects prior mucosal exposure to the bacterium, thenmost of the children are not infected by M. catarrhalis in the first fewmonths of age. One of the reasons may be that the maternal antibodiespresent in the young children-protect them from infection at this age.This is consistent with the finding that young children seldom carrythis bacterium and do not develop M. catarrhalis disease during thefirst months of life (Ejlertsen et al., 1994a).

Children may become susceptible to M. catarrhalis infection as maternalantibodies wane. In this study, the sera from 6 to 7 month old childrenhad the lowest level of IgG antibodies to the UspA proteins and barelydetectable bactericidal titers against whole cells of M. catarrhalis. By15 months of age, nearly all children had serum IgA antibodies to theUspA proteins, and the level of IgA antibodies had significantlyincreased along with the level of IgG antibodies and bactericidalactivity when compared with children of 6 to 7 months of age. Thissuggested that these children had been exposed to the bacterium andmounted an antibody response. The fifteen sera from the group of 18-36month old children all had IgG and IgA titers to the UspA proteins andthe bactericidal titers varied greatly. The UspA specific IgG antibodiesin the older children's sera had different characteristics than theantibodies from the two month old children. First, the IgG1 antibodytiter was significantly higher than the IgG3 titer in children's sera,while the opposite was true for the 2 month old children (FIG. 10).Second, most sera from 2 month old children had bactericidal activity,while bactericidal activity was barely detectable in the sera fromchildren of 6 months or older. The low antibody level and the low serumbactericidal activity seen in children between 6-36 months of age isconsistent with the epidemiological findings that children of this agegroup have the highest colonization rate and highest incidence of M.catarrhalis disease (Bluestone, 1986; Ejlertsen et al., 1994b; Leinonenet al., 1981; Roitt et al., 1985; Ruuskanen and Heikkinen, 1994; Sethiet al., 1995; Teele et al., 1989).

Adults, a population usually resistant to M. catarrhalis infections(Catlin, 1990; Ejlertsen et al., 1994a), were found to have consistentlyhigher levels of IgG antibodies to the UspA proteins as well as higherserum bactericidal activity than children. The bactericidal activity ofthe adult sera was clearly antibody-mediated since immunoglobulindepleted sera had no activity (Chen et al., 1996), and the antibodiespurified from adult plasma exhibited complement dependent bactericidalactivity. The antibodies purified from human sera using UspA1 or UspA2from a single isolate exhibited killing against multiple strains. Thisresult indicates that humans developed bactericidal antibodies towardthe conserved epitopes of UspA proteins in response to naturalinfections.

In all adult samples, the IgG antibodies were primarily of the IgG1 andIgG3 subclasses with IgG3 being higher. This is consistent with previousreports that the IgG3 subclass is a major constituent of the immuneresponse to M. catarrhalis in adults and children greater than 4 yearsof age, but not in younger children (Carson et al., 1994; Goldblatt etal., 1990). Of the four IgG subclasses in humans, IgG3 constitutes onlya minor component of the total immunoglobulin in serum. However, IgG3antibody has the highest affinity to interact with C1q, the initial stepin the classic complement pathway leading to elimination of thebacterium by both complement-dependent killing and opsono-phagocytosis(Roitt et al., 1985). Since IgG3 antibody is efficiently transferredacross the placenta, it may also confer protective immunity to infants.The data from this study indicate that IgG3 antibody to the UspAproteins is an important component of the immune response to naturalinfection and has in vitro biological activity.

As clinical information related to M. catarrhalis infection was notcollected for the study subjects, it is unknown how the antibodies toUspA1 or UspA2 were induced. When antibodies made against the UspAproteins in guinea pigs were tested for reactivity with other bacterialspecies, including Pseudomonas aeruginosa, Neisseria meningitidis,Neisseria gonorrhoeae, Bordetella pertussis, Escherichia coli, andnontypable Haemophilus influenzae by western blot, no reactivity wasdetected. This suggests that the antibodies were elicited as a specificresponse to the UspA antigens of M. catarrhalis. This is consistent withthe high colonization rate and the endemic nature of this organism inhuman populations. Since the affinity purified antibodies to the twoUspA proteins were cross-reactive, it could not be determined whetherthe human antibodies were elicited by one or both proteins. It seemedclear that the shared sequence between these two proteins was the maintarget of the bactericidal antibodies.

In summary, this study demonstrated that antibodies to the two UspAproteins are present in nearly all humans regardless of age. The overalllevel and subclass distribution of these antibodies, however, wereage-dependent. IgG antibodies against UspA1 and UspA2 werecross-reactive, and are a major source of serum bactericidal activity inadults. The level of these antibodies and serum bactericidal activityappears to correlate with age-dependent resistance to M. catarrhalisinfection. Since humans make an antibody response to many other M.catarrhalis antigens in addition to UspA1 and UspA2 after naturalinfection, it remains to be determined if immunization with one or bothUspA proteins will confer adequate protection in susceptiblepopulations.

Example VI UspA2 as a Carrier for Oligosaccharides

UspA2 as a Pneumococcal Saccharide Carrier

This study demonstrates that UspA2 can serve as a carrier for apneumococcal saccharide. A seven valent pneumococcal polysaccharide wasconjugated to UspA2 by reductive amination. Swiss Webster mice wereimmunized on wk 0 and wk 4 and a final bleed taken on wk 6. Each mousewas, immunized subcutaneously (s.c.) in the abdomen with 1 μgcarbohydrate per dose with aluminum phosphate as the adjuvant. A groupof mice was immunized with the PP7F-CRM conjugate as a control. The datafor the sera from the 6 wk bleed are shown in Table XXII, Table XXII,and Table XXIV. The conjugate elicited antibodies against both thepolysaccharide as well as bactericidal antibodies to M. catarrhalis.These results demonstrate that UspA2 can serve a carrier for elicitingantibodies to this pneumococcal saccharide and retain its immunogenicityto UspA2.

TABLE XXII Titers elicited by 7F conjugates to the pneumococcalpolysaccharide 7F Antigen IgG ELISA titer to Pn Ps 7F* PP7F-UspA2 mix<100 PP7F-UspA2 conjugate 9,514 PP7F-CRM conjugate 61,333 *Pool of serafrom five mice.

TABLE XXIII ELISA titers of sera against whole cells of three M.catarrhalis isolates Immunogen Strain Tested Group¹ 035E 430-3451230-359 PP7F-UspA2² mix 51,409 4,407 9,124 PP7F CRM conjugate 56 49 47PP7F UspA2 conjugate 31,111 3,529 8,310 ¹Vaccine group consists of 5Swiss-Webster mice. Each group immunized at wk 0 and wk 3 and serumcollected at wk 6. ²Vaccine composed of 1 μg Pneumo Type 7F and 1 μgUspA2 adjuvanted with aluminum phosphate.

TABLE XXIV Complement dependent bactericidal antibodies against three M.catarrhalis isolates Immunogen Strain Tested Group¹ 035E 430-3451230-359 PP7F-UspA2 mix 400 400 400 PP7F CRM conjugate <100 <100 <100PP7F UspA2 conjugate 400 400 200 ¹BC₅₀ titer is highest serum dilutionat which >50% of bacteria were killed as compared to serum from wk 0mice. The most concentrated serum tested was a 1:100 dilution.UspA2 as an Haemophilus b Oligosaccharide Carrier.

This study demonstrates that UspA2 can serve as a carrier for anHaemophilus influenzae type b oligosaccharide (HbO). An HbO sample(average DP=24) was conjugated to UspA2 by aqueous reductive aminationin the presence of 0.1% Triton X-100. The ratio of the HbO to UspA2 was2:1 by weight. Conjugation was allowed to proceed for 3 days at 35° C.and the conjugate diafiltered using an Amicon 100K cutoff membrane. Theconjugate ratio (mg carbohydrate/mg UspA2) was 0.43:1. The carbohydratewas determined by orcinal assay and the protein by Lowry. The number ofhydroxy-ethyl lysines was determined by amino acid analysis and found tobe 12.6.

The immunogenicity of the conjugate was examined by immunizingSwiss-Webster mice. The mice were immunized twice on wk 0 and wk 4 with1 μg of carbohydrate. No adjuvant was used with the conjugate, but wasused with UspA2. The sera were pooled and titered. The reactivity towardHbPS by the radioantigen binding assay (RABA) was similar to that seenwhen HbO is conjugated to CRM₁₉₇ (Table XXV). The whole cell titertoward the homologous M. catarrhalis isolate (O35E) was similar to thatseen for non-conjugated USpA2 (Table XXVI), as were the bactericidaltiters (Table XXVII). Thus, when a carbohydrate antigen that typicallyelicits a RABA titer less than 0.10 is conjugated to UspA2, it becomesimmunogenic.

TABLE XXV Comparison of immunogenicity of HbO conjugated to UspA2 to HbOconjugated to CRM₁₉₇ to Haemophilus b polysaccharide by RadioantigenBinding Assay (RABA) Week HbO-CRM₁₉₇ Hbo-UspA2 0 <0.10 <0.10 3 2.51 2.874 4.46 3.56 6 58.66 18.92

TABLE XXVI Comparison of immunogenicity of HbO-UspA2 conjugate with non-conjugated UspA2 by ELISA against whole cell of the O35E isolate to M.catarrhalis Week UspA2^(a) Hbo-UspA2 0 <50 <50 4 54,284 17,424 6 345,057561,513 ^(a)5 μg UspA2 adjuvanted with 500 μg aluminum phosphate.

TABLE XXVII Bactericidal of sera toward two M. catarrhalis isolates.Isolate UspA2^(a) Hbo-UspA2 O35E 4,500 >4,500 345 n.d. 450 ^(a)5 μgUspA2 adjuvanted with 500 μg aluminum phosphate. n.d. = not determined

Example VII Association of Mouse Serum Sensitivity with Expression ofMutant Forms of UspA2

When bacteria are killed in the presence of serum that lack specificantibodies toward them, it is called “serum sensitivity.” In the case ofM. catarrhalis, the mutants lacking an intact UspA2 protein have beenfound to be serum sensitive. These mutants were constructed so that one(O35E.1; refer to Example IX for a description of isolates O35E.1,O35E.2 and O35E.12) did not express UspA1, one (O35E.2) did not expressUspA2, and one (O35E.12) did not express either protein based on a lackof reactivity with the 17C7 monoclonal antibody. The O35E.2 and O35E.12,however, expressed a smaller truncated form UspA2 (tUspA2) that reactswith antibodies prepared by immunizing mice with purified UspA2. ThetUspA2 could be detected in a western blot of bacterial lysates usingeither polyclonal anti-UspA2 sera or the MAb 13-1. The size of thesmaller form was consistent with the gene truncation used for theconstruction of the two mutants.

This bactericidal capacity was tested by mixing the non-immune mousesera, a 1:5 dilution of human complement and a suspension of bacteria(Approx. 1000 cfu) in the wells of a microtiter plate. The mouse serawere tested at both a 1:50 and 1:100 dilution. The number of survivingbacteria was then determined by spreading a dilution of this bacterialsuspension on agar growth medium. The killing was considered significantwhen fewer than 50% viable bacteria as cfu's were recovered relative tothe samples without mouse sera. Killing by the non-immune sera was seenonly for the mutants lacking a “complete” UspA2 (Table XXVIII).

TABLE XXVIII Bactericidal activity of the pre-immune sera from Balb/cmice Bactericidal Activity of Mutant Proteins Expressed Normal MouseSera 035E UspA1 & UspA2 − 035E.1 UspA2 − O35E.2 UspA1 & tUspA2 + 035E.12tUspA2 +

Example VIII Identification of a Decapeptide Epitope in UspA1 that BindsMAb 17C7

It was clear from the work with different strains of M. catarrhalis andanalyses of their protein sequences of UspA1 that certain epitopicregions must exist which are similar, if not identical, in all of thestrains and provide the basis of the immunogenic response in humans. Inorder to identify such immunogenic epitope(s), peptides spanning theUspA1 region known to contain the binding site for MAb 17C7 wereprepared and examined for their ability to bind to MAb 17C7.

Specifically, overlapping synthetic decapeptides, as shown in Table XXIXand FIG. 12, that were N-terminally bound to a membrane composed ofderivatized cellulose were obtained from Research Genetics Inc.(Huntsville, Ala.). After five washes with PBS-Tween containing 5% (w/v)non-fat dry milk, the membrane was subsequently incubated with MAb 17C7(in the form of hybridoma culture supernatant) overnight at 4° C.Following three washes with PBS-Tween, the membrane was incubatedovernight at 4° C. with gentle rocking with 10⁶ cpm of radioiodinated(specific activity 2×10⁷ cpm/μg protein), affinity-purified goatanti-mouse immunoglobulin. The membrane was then washed as before andexposed to X-ray film Fuji RX safety film, Fuji Industries, Tokyo,Japan).

TABLE XXIX Decapeptides Used to Identify Binding Site for MAb 17C7PEPTIDE # PEPTIDE SEQUENCE 9 SGRLLDQKAD SEQ ID NO:81 10 QKADIDNNIN SEQID NO:82 11 NNINNIYELA SEQ ID NO:83 12 NNIYELAQQQ SEQ ID NO:84 13YELAQQQDQH SEQ ID NO:18 14 AQQQDQHSSD SEQ ID NO:85 15 QDQHSSDIKT SEQ IDNO:86 16 HSSDIKTLKN SEQ ID NO:87 17 DIKTLKNNVE SEQ ID NO:88 18TLKNNVEEGL SEQ ID NO:89 19 EEGLLDLSGR SEQ ID NO:90 20 LSGRLIDQKA SEQ IDNO:91 21 DQKADIAKNQ SEQ ID NO:92 22 AKNQADIAQN SEQ ID NO:93 23IAQNQTDIQD SEQ ID NO:94 24 DIQDLAAYNE SEQ ID NO:95

It is clear from the dot blot results shown in the autoradiograph (FIG.13) that peptide 13, YELAQQQDQH (SEQ ID NO:18) exhibited optimal bindingof MAb 17C7 with peptide 14 (SEQ ID NO:85) exhibiting less than optimalbinding. This same peptide (SEQ ID NO:18) is present in UspA2 whichexplains why both proteins bind to MAb 17C7.

Interestingly, peptide 12 shows no binding and binding by peptides 15,16, 19, 22, 23 is probably non-specific. Thus, a comparison of peptides12, 13, and 14 yields the conclusion that the 7-mer AQQQDQH (SEQ IDNO:17) is an essential epitope for MAb 17C7 to bind to UspA1 and UspA2.This conclusion is in agreement with the current understanding that animmunogenic epitope may comprise as few as five, six or seven amino acidresidues.

Example IX Phenotypic Effect of Isogenic UspA1 and UspA2 Mutations on M.catarrhalis Strain-O35E

Materials and Methods

Bacterial strains, plasmids and growth conditions. The bacterial stainsand plasmids used in this study are listed in Table XXX. M. catarrhalisstrains were routinely grown at 37° C. on Brain-Heart Infusion (BHI)agar plates (Difco Laboratories, Detroit, Mich.) in an atmosphere of 95%air-5% CO₂ supplemented, when necessary, with kanamycin (20 μg/ml)(Sigma Chemicals Co.; St. Louis, Mo.) or chloramphenicol (0.5 μg/ml)(Sigma), or in BHI broth. The BHI broth used to grow M. catarrhaliscells for attachment assays was sterilized by filtration. Escherichiacoli strains were cultured on Luria-Bertani (LB) agar plates (Maniatiset al., 1982) supplemented, when necessary, with ampicillin (100 μg/ml),kanamycin (30 μg/ml), or chloramphenicol (30 μg/ml).

TABLE XXX Bacterial Strains and Plasmids Used in this Study Strain orplasmid Description Source or reference M. catarrhalis 035E Wild-typeisolate from Helminen et al., 1994 middle ear fluid O35E.1 Isogenicmutant of O35E Aebi et al., 1997 with a kan cartridge in the uspA1structural gene O35E.2 Isogenic mutant of O35E Aebi et al., 1997 with akan cartridge in the uspA2 structural gene O35E.12 Isogenic mutant ofO35E This study with a kan cartridge in the uspA2 structural gene and acat cartridge in the uspA1 structural gene P-44 Wild-type isolate thatSoto-Hernandez et al., exhibits rapid 1989 hemagglutination P-48Wild-type isolate that Soto-Hernandez et al., exhibits slow 1989hemagglutination Escherichia coli DH5α Host for cloning studiesStratagene Plasmids pBluescript II Cloning vector; Amp^(r) StratagenepUSPA1 pBluescript II SK+ with a Aebi et al., 1997 2.7 kb insertcontaining most of the uspA1 gene of M. catarrhalis strain O35EpUSPA1CAT pUSPA1 with a cat cartridge This study replacing the 0.6 kbBglII fragment of the uspA1 gene

Characterization of outer membrane proteins. Whole cell lysates andouter membrane vesicles of M. catarrhalis strains were prepared asdescribed (Murphy and Loeb, 1989; Patrick et al., 1987). Proteinspresent in these preparations were resolved by SDS-PAGE and detected bystaining with Coomassie blue or by western blot analysis as described(Helminen et al., 1993a).

Monoclonal antibodies (MAbs). MAb 17C7, a murine IgG antibody thatreacts with a conserved epitope of both UspA1 and UspA2 from M.catarrhalis strain O35E, as described in earlier examples herein, wasused for immunologic detection of these proteins. MAb 17C7 was used inthe form of hybridoma culture supernatant fluid in western blot analysisand in the indirect antibody-accessibility assay. MAb 3F12, an IgG MAbspecific for the major outer membrane protein of Haemophilus ducreyi(Klesney-Tait et al., 1997), was used as a negative control in theindirect antibody-accessibility assay.

Molecular cloning methods. Chromosomal DNA of M. catarrhalis strain O35Ewas used as the template in a polymerase chain reaction (PCR™) systemtogether with oligonucleotide primers derived from either just after thestart of the strain O35E uspA1 open reading frame (i.e., P1 in FIG. 14)or just after the end of this open reading frame (i.e., P2 in FIG. 14).These primers were designed to contain a BamHI restriction site at their5′-end. The sequence of these primers was:

(SEQ ID NO:96) P1-5′-CGGGATCCGTGAAGAAAAATGCCGCAGGT-3′; (SEQ ID NO:97)P2-5′-CGGGATCCCGTCGCAAGCCGATTG-3′.DNA fragments were amplified using a PTC 100 Programmable ThermalController (MJ Research, Inc., Cambridge, Mass.) and the GeneAmp PCR™kit (Roche Molecular Systems, Inc., Branchburg, N.J.). PCR™ productswere extracted from 0.7% agarose gel slices using the Qiaex GelExtraction Kit (Qiagen, Inc., Chadsworth, Calif.) and digested withBamHI (New England Biolabs, Inc., Beverly, Mass.) for subsequentligation into the BamHI site of pBluescript II SK+ (Stratagene, LaJolla, Calif.). Ligation reactions were performed with overnightincubation at 16° C. using T4 DNA ligase (Gibco BRL, Inc., Gaithersburg,Md.). Competent E. coli DH5α cells were transformed with the ligationreaction mixture according to a standard heat-shock procedure (Sambrooket al., 1989) and the desired recombinants were selected by culturing inthe presence of an appropriate antimicrobial compound. The 1.3 kbchloramphenicol (cat) resistance cartridge was prepared by excision(using BamHI) from pUCΔECAT (Wyeth-Lederle, Rochester, N.Y.). The catcartridge was subsequently ligated into BglII restriction sites locatedin the mid-portion of cloned segment from the uspA1 gene and, aftertransformation of competent E. coli DH5 cells, recombinant clones wereidentified by selection on solidified media containing chloramphenicol.

Transformation of M. catarrhalis. The electroporation method used fortransformation of M. catarrhalis strain O35E has been described indetail (Helminen et al., 1993b). Briefly, a 30-ml portion of alogarithmic-phase broth culture (10⁹ colony forming units [cfu]/ml) washarvested by centrifugation, washed three times with 10% (v/v) glycerolin distilled water, and resuspended in 100 μl of the same solution. A20-μl portion of these cells was electroporated with 5 μg of linear DNA(i.e., the truncated uspA1 gene containing the cat cartridge) in 5 μl ofwater in a microelectroporation chamber (Cel-Porator Electroporationsystem; Bethesda Research Laboratories, Gaithersburg, Md.) by applying afield strength of 16.2 kV over a distance of 0.15 cm. Followingelectroporation, the cell suspension was transferred to 1 ml of BHIbroth and incubated with shaking at 37° C. for 90 min. Ten 100-μlportions were then spread on BHI agar plates containing the appropriateantimicrobial compound.

Southern blot analysis. Chromosomal DNA purified from wild-type andmutant M. catarrhalis strains strains was digested with either PvuII orHindIII (New England Biolabs) and Southern blot analysis was performedas described (Sambrook et al., 1989). Double-stranded DNA probes werelabeled with ³²P by using the Random Primed DNA Labeling Kit(Boehringer-Mannheim, Indianapolis, Ind.).

Indirect antibody-accessibility assay. Overnight BHI broth cultures ofM. catarrhalis strain O35E and its isogenic mutants were diluted in PBSbuffer containing 10% (v/v) fetal bovine serum and 0.025% (w/v) sodiumazide (PBS-FBS-A) to density of 110 Klett units (ca. 10⁹ cfu/ml) asmeasured with a Klett-Summerson colorimeter (Klett Manufacturing Co.,New York, N.Y.). Portions (100 μl) of this suspension were added to 1 mlof MAb 17C7 or MAb 3F12 culture supernatant. After incubation at 4° C.for one hour with gentle agitation, the bacterial cells were washed onceand suspended in 1 ml of PBS-FBS-A. Affinity-purified goat anti-mouseimmunoglobulin, radiolabeled with ¹²⁵I to a specific activity of 10⁸ cpmper μg, was added and the mixture was incubated for one hour at 4° C.with gentle agitation. The cells were then washed four times with 1 mlof PBS-FBS-A, suspended in 500 μl of triple detergent (Helminen et al.,1993a) and transferred to glass tubes. The radioactivity present in eachsample was measured by using a gamma counter.

Autoagglutination and hemagglutination assays. The ability of M.catarrhalis strains to autoagglutinate was assessed using bacterialcells grown overnight on a BHI agar plate. These cells were resuspendedin PBS to a turbidity of 400 Klett units in a glass tube andsubsequently allowed to stand at room temperature for ten minutes atwhich time the turbidity of this suspension was again determined. Rapidand slow autoagglutination were defined as turbidities of less that andgreater than 200 Klett units, respectively, after 10 minutes. Thehemagglutination slide assay using heparinized human group O Rh⁺erythrocytes was performed as previously described (Soto-Hernandez etal., 1989).

Serum bactericidal assay. Complement-sufficient normal adult human serumwas prepared by standard methods. Complement inactivation was achievedby heating the serum for 30 min at 56° C. A M. catarrhalis broth culturein early logarithmic phase was diluted in Veronal-buffered salinecontaining 0.10% (w/v) gelatin (GVBS) to a concentration of 1-2×10⁵cfu/ml, and 20 μl portions were added to 20 μl of native orbeat-inactivated normal human serum together with 160 μl ofVeronal-buffered saline containing 5 mM MgCl₂ and 1.5 mM CaCl₂. Thismixture was incubated at 37° C. in a stationary water bath. At time 0and at 15 and 30 min, 10 μl aliquots were removed, suspended in 75 μl ofBHI broth and spread onto prewarmed BHI agar plates.

Adherence assay. A method used to measure adherence of Haemophilusinfluenzae to Chang conjunctival cells in vitro (St Geme III and Falkow,1990) was adapted for use with M. catarrhalis. Briefly, 2-3×10⁵ HEp-2cells (ATCC CCL 23) or Chang conjunctival cells (ATCC CCL 20.2) wereseeded into each well in a 24-well tissue culture plate (Corning-Costar)and incubated for 24 h before use. A 0.3 ml volume from anantibiotic-free overnight culture of M. catarrhalis was inoculated into10 ml of fresh BHI medium lacking antibiotics and this culture wassubsequently allowed to grow to a concentration of approximately 5×10⁸cfu/ml (120 Klett units) with shaking in a gyrotory water bath. Theculture was harvested by centrifugation at 6,000×g at 4-8° C. for 10min. The supernatant was discarded and a Pasteur pipet was used togently resuspend the bacterial cells in 5 ml of pH 7.4phosphate-buffered saline (PBS) or PBS containing 015% (w/v) gelatin(PBS-G). The bacterial cells were centrifuged again and this finalpellet was gently resuspended in 6-8 ml of PBS or PBS-G.

Portions (25 μl) of this suspension (10⁷ CFU) were inoculated into thewells of a 24 well tissue culture plate containing monolayers of HEp-2or Chang cells. These tissue culture plates were centrifuged for 5 minat 165×g and then incubated for 30 min at 37° C. Non-adherent bacteriawere removed by rinsing the wells gently five times with PBS or PBS-G,and the epithelial cells were then released from the plastic support byadding 200 μl of PBS containing 0.05% trypsin and 0.02% EDTA. This cellsuspension was serially diluted in PBS or PBS-G and spread onto BIBplates to determine the number of viable M. catarrhalis present.Adherence was expressed as the percentage of bacteria attached to thehuman cells relative to the original inoculum added to the well.

Results

Construction of an isogenic M. catarrhalis mutant lacking expression ofboth UspA1 and UspA2. Construction of M. catarrhalis mutants lacking theability to express either UspA1 (mutant strain O35E.1) or UspA2 (mutantstrain O35E.2) has been described in previous examples (Aebi et al.,1997). For constructing a double mutant that lacked expression of bothUspA1 and UspA2, the 0.6 kb BglII fragment of pUSPA1 (FIG. 14A) wasreplaced by a cat cassette, yielding the recombinant plasmid pUSPA1CAT.Using the primers P1 and P2, the 3.2 kb insert of pUSPA1CAT wasamplified by PCR™. This PCR™ product was used to electroporate thekanamycin-resistant uspA2 strain O35E.2 and yielded the chloramphenicol-and kanamycin-resistant transformant O35E.12, a putative uspA1 uspA2double mutant.

Southern blot analysis was used to confirm that strains O35E.1, O35E.2,and O35E.12 were isogenic mutants and that allelic exchange had occurredproperly, resulting in replacement of the wild-type uspA1 or uspA2 gene,or both, with the mutated allele. Chromosomal DNA preparations from thewild-type parent strain O35E, the uspA1 mutant O35E.1, the uspA2 mutantO35E.2, and the putative uspA1 uspA2 mutant strain O35E.12 were digestedto completion with PvuII and probed in Southern blot analysis with DNAfragments derived from these two M. catarrhalis genes or with the kancartridge. For probing with the cat cartridge, chromosomal DNA fromstrain O35E-12 was digested with HindIII.

The uspA1-specific DNA probe was obtained by PCR™-based amplification ofM. catarrhalis strain O35E chromosomal DNA using the primers P3 and P4(FIG. 14A). A 500-bp uspA2-specific DNA fragment was amplified from O35Echromosomal DNA by PCR™ with the primers P5 and P6 (FIG. 14B). Use ofthese two gene-specific probes together with the kan and cat cartridgesin Southern blot analysis confirmed that strain O35E.12 was a uspA1uspA2 double mutant.

Characterization of selected proteins expressed by the wild-type andmutant M. catarrhalis strains. Proteins present in outer membranevesicles extracted from the wild-type and these three mutant strainswere resolved by SDS-PAGE and either stained with Coomassie blue (FIG.15A) or probed with MAb 17C7 in western blot analysis (FIG. 15B). Thewild-type parent strain O35E possessed a very high molecular weight banddetectable by Coomassie blue staining (FIG. 15A, lane 1, closed arrow)that was also similarly abundant in the uspA1 mutant O35E.1 (FIG. 15A,lane 2). The uspA2 mutant O35E.2 (FIG. 15A, lane 3) had a much reducedlevel of expression of a band in this same region of the gel; this bandwas not visible at all in the uspA1 uspA2 double mutant O35E.12 (FIG. 2,panel A, lane 4).

Western blot analysis revealed that the wild-type strain (FIG. 15B,lane 1) expressed abundant amounts of MAb 17C7-reactive antigen, most ofwhich had a very high molecular weight, in excess of 220,000. Thewild-type strain also exhibited discrete antigens with apparentmolecular weights of approximately 120,000 and 85,000 which bound thisMAb (FIG. 15B, lane 1, open and closed arrows, respectively). The uspA1mutant O35E.1 (FIG. 15B, lane 2) lacked expression of the 120 kDaantigen, which was proposed to be the monomeric form of UspA1, but stillexpressed the 85 kDa antigen. The amount of very high molecular weightMAb 17C7-reactive antigen expressed by this uspA1 mutant appeared to beequivalent to that expressed by the wild-type strain. The uspA2 mutantO35E.2 (FIG. 15B, lane 3) expressed the 120 kDa antigen but lackedexpression of the 85 kDa antigen which was proposed to be the monomericform of the UspA2 protein. In contrast to the uspA1 mutant, the uspA2mutant had relatively little very high molecular weight antigen reactivewith MAb 17C7. Finally, the uspA1 uspA2 double mutant O35E.12 (FIG. 15B,lane 4) expressed no detectable MAb 17C7-reactive antigens.

Binding of MAb 17C7 to whole cells of the wild-tyre and mutant strains.The indirect antibody-accessibility assay was used to determine whetherboth UspA1 and UspA2 are exposed on the surface of M. catarrhalis andaccessible to antibody. Whole cells of both the wild-type strain O35Eand the uspA1 mutant O35E.1 bound similar amounts of MAb 17C7 (TableXXXI). This result suggested that UspA2 is expressed on the surface ofM. catarrhalis, or at least on the surface of the uspA1 mutant. TheuspA2 mutant O35E.2 bound substantially less MAb 17C7 than did thewild-type strain, but the level of binding was still at least an orderof magnitude greater than that obtained with an irrelevant IgG Mabdirected against a H. ducreyi outer membrane protein (Table XXXI). Asexpected from the western blot analysis, the uspA1 uspA2 double mutantO35E.12 did not bind MAb 17C7 at a level greater than obtained with thenegative controls involving the H. ducreyi-specific MAb (Table XXXI).

TABLE XXXI Binding of MAb 17C7 to the Surface of Wild-Type and MutantStrains of M. catarrhalis Bindings^(a) of Strain MAb 17C7 MAb 3F12b O35E(wild-type)  145,583^(c) 4,924 O35E.1 (uspA1 mutant) 154,119 4,208O35E.2 (uspA2 mutant)  96,721 4,455 O35E.12 (uspA1 uspA2 double mutant) 6,081 3,997 ^(a)Counts per min of ¹²⁵I-labeled goat anti-mouseimmunoglobulin bound to MAbs attached to the bacterial cell surface, asdetermined in the indirect antibody-accessibility assay. ^(b)MAb 3F12, amurine IgG antibody specific for a H. ducreyi outer membrane protein(Klesney-Tait et al., 1997), was included as a negative control. ^(c)Thevalues represent the mean of two independent studies.

Characterization of the growth, autoagglutination, and hemagglutinationproperties of the wild-type and mutant strains. The colony morphology ofthese three mutant strains grown on BHI agar plates did not differ fromthat of the wild-type strain parent strain. Similarly, the rate andextent of growth of all four of these strains in BHI broth were verysimilar if not identical (FIG. 16). In an autoagglutination assayperformed as described in above in the Materials and Methods section ofthis example, all four strains exhibited the same rate ofautoagglutination. Finally, there was no detectable difference betweenthe wild-type parent and the three mutants in a hemagglutination assayusing human group O erythrocytes (Soto-Hernandez et al., 1989). Controlhemagglutination studies were performed using a pair of M. catarrhalisisolates (i.e., strains P-44 and P48) previously characterized as havingrapid or slow rates, respectively, of hemagglutination (Soto-Hernandezet al., 1989).

Effect of the uspA1 and uspA2 mutations on the ability of M. catarrhalisto adhere to human cells. Preliminary studies revealed that thewild-type M. catarrhalis strain O35E adhered readily to HeLa cells,HEp-2 cells, and Chang conjunctival cells in vitro. To determine whetherlack of expression of UspA1 or UspA2 affected this adherence ability,the wild-type and the three mutant strains were first used in anattachment assay with HEp-2 cells. In this set of studies, PBS was usedas the diluent for washing the HEp-2 cell monolayers and for serialdilution of the trysinized HEp-2 cell monolayer at the completion of theassay. Both the wild-type strain and the uspA2 mutant O35E.2 exhibitedsimilar levels of attachment to HEp-2 monolayers (Table XXXI). The uspA1mutant O35E.1, however, was less able to adhere to these HEp-2 cells;lack of expression of UspA1 reduced the level of attachment byapproximately six-fold (Table XXXII). The uspA1 uspA2 double mutantO35E.12 exhibited a similarly reduced level of attachment (Table XXXII).

TABLE XXXII Adherence of Wild-Type and Mutant Strains of M. catarrhalisto HEp-2 and Chang Conjunctival Cells in vitro Adherence^(a) to StrainHEp-2 cells^(b) Chang cells^(c) O35E (wild-type) 14.7 ± 4.9  51.4 ± 30.8O35E.1 (uspA1 mutant) 2.4 ± 0.9 (0.006^(d)) 0.8 ± 0.5 (0.002^(d)) O35E.2(uspA2 mutant) 19.1 ± 7.0  (0.213^(d)) 55.9 ± 16.7 (0.728^(d)) O35E.12(uspA1 uspA2 2.3 ± 1.8 (0.011^(d)) 0.6 ± 0.2 (0.002^(d)) double mutant)^(a)Adherence is expressed as the percentage of the original inoculumthat was adherent to the human epithelial cells at the end of the 30 minincubation period. Each number represents the mean (± S.D.) of twoindependent studies. ^(b)PBS was used for washing of the monolayers andfor serial dilutions of adherent M. catarrhalis. ^(c)PBS-G was used forwashing of the monolayers and for serial dilutions of adherent M.catarrhalis. ^(d)P value when compared to the wild-type strain O35Eusing the two-tailed Student t-test.

Control studies revealed, however, that M. catarrhalis cells did notsurvive well in the PBS used for washing of the HEp-2 monolayer andserial dilution of the attached M. catarrhalis organisms. When 10⁸ CFUof the wild-type and mutant M. catarrhalis strains were suspended inPBS, serially diluted, and allowed to stand for 30 min on ice, theviable number of bacteria decreased to 10⁷ CFU. In contrast, when PBScontaining 0.15% (w/v) gelatin (PBS-G) was used for this same type ofexperiment, there was no reduction in the viability of these M.catarrhalis stains over the duration of the experiment. When the HEp-2cell-based attachment studies were repeated using PBS-G for washing theHEp-2 cell monolayer and as the diluent, there was only a three-foldreduction in adherence of the uspA1 mutant relative to that obtainedwith the wild-type parent strain. This finding suggested that theoriginal six-fold difference in attachment ability observed between thewild-type and uspA1 mutant stain may have been attributable in part toviability problems caused by the use of the PBS wash and diluent.

Subsequent studies using Chang conjunctival cells as the target forbacterial attachment together with a PBS-G wash and diluent revealed asubstantial difference in the attachment abilities of the wild-typestrain and the uspA1 mutant (Table XXXII). Whereas the wild-type anduspA2 mutant exhibited similar levels of attachment to the Chang cells,the extent of attachment of the uspA1 mutant was nearly two orders ofmagnitude less than that of the wild-type parent strain. The uspA1 uspA2double mutant also exhibited a much reduced level of attachment similarto obtained with the uspA1 mutant (Table XXXII).

Effect of the uspA1 and uspA2 mutations on serum resistance of M.catarrhalis. Similar to the majority of disease isolates of M.catarrhalis (Hol et al., 1993; 1995; Verduin et al., 1994), thewild-type strain O35E was resistant to killing by normal human serum invitro (Helminen et al., 1993b). To examine the effect of the lack ofexpression of UspA1 or UspA2 on serum resistance, the wild-type strainand the three mutant strains were tested in a serum bactericidal assay.Both the wild-type strain (FIG. 17, closed diamonds) and the uspA1mutant O35E.1 (FIG. 17, closed triangles) were able to grow in thepresence of normal human serum, indicating that lack of expression ofUspA1 did not adversely affect the ability of strain O35E.1 to resistkilling by normal human serum. However, both the uspA2 mutant O35E.2(FIG. 17, closed circles) and the uspA1 uspA2 double mutant O35E.12(FIG. 17, closed squares), having in common the lack of expression ofUspA2, were readily killed by normal human serum. Heat-basedinactivation of the complement system present in this normal human serumeliminated the ability of this serum to kill these latter two mutants(FIG. 17, open circles and squares).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An isolated nucleic acid encoding the UspA2 antigen (SEQ ID NO:11) ofthe M. catarrhalis isolate TTA24.
 2. An isolated nucleic acid having theuspA2 DNA sequence (SEQ ID NO:12) of the M. catarrhalis isolate TTA24.